Table 1_Identification and validation of novel risk genes for intervertebral disc disorder by integrating large-scale multi-omics analyses and experimental studies.xlsx

IntroductionAlthough genome-wide association studies (GWAS) have identified multiple genetic loci linked to intervertebral disc disorder (IDD), their functional characterization remains largely unelucidated. We aim to leverage an integrative analytical pipeline to identify novel IDD risk genes from genetic associations and experimentally validate their functional roles.
MethodsWe integrated transcriptome-wide association studies (TWAS), proteome-wide association studies (PWAS), expression and protein quantitative trait loci (eQTL and pQTL) colocalization analyses to identify potential causal genes for IDD. Enrichment analysis, expression profiling, protein-protein interaction (PPI) network construction, and druggability evaluation were also performed for the prioritized causal candidates. Subsequently, human intervertebral disc (IVD) tissues spanning degeneration grades and an in vivo mouse IDD model were employed to functionally characterize candidate risk genes.
ResultsIntegrative analysis of TWAS and PWAS with colocalization studies identified 104 genes and 10 proteins exhibiting causal associations with IDD. The identified genes/proteins were enriched in extracellular matrix organization, cellular senescence and collagen formation. Crucially, TMEM190, CILP2, and FOXO3 were demonstrated consistent evidence across TWAS, two independent PWAS datasets, and corresponding colocalization analyses, with CILP2 emerging as a potentially druggable target. Differential expression analysis revealed significant upregulated TMEM190 and CILP2, along with downregulated FOXO3 during IVD degeneration. These results were subsequently confirmed at protein levels in clinical specimens. Mouse model experiments further established that down-regulation of CILP2 alleviated IDD progression.
DiscussionCollectively, this work provides an updated compendium of putative IDD risk genes and delineates pathogenic roles for TMEM190, CILP2, and FOXO3, providing a broad hint for further research on novel mechanism and therapeutic targets for IDD.

引言
尽管全基因组关联分析（Genome-Wide Association Studies, GWAS）已筛选出多个与椎间盘退变疾病（Intervertebral Disc Disorder, IDD）相关的遗传位点，但其功能表征仍未得到充分阐明。本研究旨在借助整合分析流程，从遗传关联数据中挖掘新型IDD风险基因，并通过实验验证其功能作用。

方法
本研究整合了转录组全关联分析（Transcriptome-Wide Association Studies, TWAS）、蛋白质组全关联分析（Proteome-Wide Association Studies, PWAS）以及表达数量性状位点、蛋白质数量性状位点共定位分析（Expression and Protein Quantitative Trait Loci, eQTL and pQTL），以筛选IDD潜在的因果基因。同时对筛选出的优先因果候选基因开展富集分析、表达谱分析、蛋白质相互作用（Protein-Protein Interaction, PPI）网络构建以及可药性评估。随后，本研究使用覆盖不同退变分级的人体椎间盘（Intervertebral Disc, IVD）组织以及体内小鼠IDD模型，对候选风险基因进行功能表征。

结果
整合TWAS、PWAS及共定位分析的结果，共筛选出104个基因与10个蛋白质与IDD存在因果关联。上述筛选出的基因/蛋白质显著富集于细胞外基质组织、细胞衰老及胶原形成通路中。尤为关键的是，TMEM190、CILP2与FOXO3在TWAS、两个独立的PWAS数据集以及对应的共定位分析中均呈现出一致的关联证据，其中CILP2被鉴定为潜在的可药性靶点。差异表达分析显示，在椎间盘退变过程中，TMEM190与CILP2表达显著上调，而FOXO3表达显著下调。上述结果随后在临床标本的蛋白质水平得到了验证。小鼠模型实验进一步证实，下调CILP2的表达可缓解IDD的疾病进展。

讨论
综上，本研究更新了推定的IDD风险基因目录，并阐明了TMEM190、CILP2与FOXO3的致病作用，为后续IDD新型发病机制研究及治疗靶点开发提供了重要指引。