Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such as reporter assays and genome editing. In this report, we undertook such studies on the osteoarthritis (OA) risk that is marked by single nucleotide polymorphism (SNP) rs34195470 (A > G). The OA risk-conferring G allele of this SNP associates with increased DNA methylation (DNAm) at two CpG dinucleotides within WWP2. This gene encodes a ubiquitin ligase and is the host gene of microRNA-140 (miR-140). WWP2 and miR-140 are both regulators of TGFβ signaling. Nucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNAm quantified by pyrosequencing at the two CpGs plus 14 flanking CpGs. CpGs were tested for transcriptional regulatory effects using a chondrocyte cell line and reporter gene assay. DNAm was altered using epigenetic editing, with the impact on gene expression determined using RT-qPCR. In silico analysis complemented laboratory experiments. rs34195470 genotype associates with differential methylation at 14 of the 16 CpGs in OA cartilage, forming a methylation quantitative trait locus (mQTL). The mQTL is less pronounced in foetal cartilage (5/16 CpGs). The reporter assay revealed that the CpGs reside within a transcriptional regulator. Epigenetic editing to increase their DNAm resulted in altered expression of the full-length and N-terminal transcript isoforms of WWP2. No changes in expression were observed for the C-terminal isoform of WWP2 or for miR-140. As far as we are aware, this is the first experimental demonstration of an OA association signal targeting specific transcript isoforms of a gene. The WWP2 isoforms encode proteins with varying substrate specificities for the components of the TGFβ signaling pathway. Future analysis should focus on the substrates regulated by the two WWP2 isoforms that are the targets of this genetic risk.

中文翻译：

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泛素连接酶基因 WWP2 的特定异构体通过差异甲基化 DNA 序列成为骨关节炎遗传风险的目标

从遗传关联信号到效应基因和可靶向分子机制的转变需要应用功能性精细作图工具，例如报告分析和基因组编辑。在本报告中，我们对以单核苷酸多态性（SNP）rs34195470（A > G）为标志的骨关节炎（OA）风险进行了此类研究。该 SNP 的 OA 风险 G 等位基因与 WWP2 内两个 CpG 二核苷酸处 DNA 甲基化 (DNAm) 的增加相关。该基因编码泛素连接酶，是 microRNA-140 (miR-140) 的宿主基因。 WWP2 和 miR-140 都是 TGFβ 信号传导的调节因子。从成人 OA（关节置换术）和胎儿软骨中提取核酸。对样品进行基因分型，并通过焦磷酸测序对两个 CpG 和 14 个侧翼 CpG 进行 DNAm 定量。使用软骨细胞系和报告基因测定来测试 CpG 的转录调节作用。使用表观遗传编辑改变 DNAm，并使用 RT-qPCR 确定对基因表达的影响。计算机分析补充了实验室实验。 rs34195470 基因型与 OA 软骨中 16 个 CpG 中的 14 个的差异甲基化相关，形成甲基化数量性状基因座 (mQTL)。 mQTL 在胎儿软骨中不太明显 (5/16 CpG)。报告基因检测显示 CpG 位于转录调节因子内。增加 DNAm 的表观遗传编辑导致 WWP2 全长和 N 末端转录亚型的表达发生改变。没有观察到 WWP2 的 C 端亚型或 miR-140 的表达变化。据我们所知，这是针对基因特定转录亚型的 OA 关联信号的首次实验演示。 WWP2 亚型编码的蛋白质对 TGFβ 信号通路的成分具有不同的底物特异性。未来的分析应重点关注受两种 WWP2 同工型调节的底物，这两种亚型是这种遗传风险的目标。