Integrative Mendelian Randomization and Proteomic Analyses Reveal Genetically Supported Candidate Causal Biomarkers for Non-Traumatic Osteonecrosis of the Femoral Head

Abstract

INTRODUCTION: Non-traumatic osteonecrosis of the femoral head (NONFH) is a highly disabling orthopedic disorder characterized by an insidious onset and significant clinical challenges. Although quantitative proteomic profiling of necrotic and adjacent healthy bone tissues has elucidated aspects of disease mechanisms, fundamental limitations remain in comprehensively understanding NONFH pathogenesis. OBJECTIVES: This study aimed to identify causal biomarkers for NONFH by integrating differentially expressed proteins (DEPs) with transcriptome-wide association study (TWAS) and Mendelian randomization (MR) analyses. METHODS: Data-independent acquisition (DIA)-based proteomics was performed on necrotic, sclerotic, and healthy bone tissues to identify DEPs. TWAS-prioritized genes were subjected to MR using blood and musculoskeletal tissue-specific expression quantitative trait loci (eQTLs). Reliable causal relationships were retained by excluding pleiotropy or heterogeneity and requiring consistent directions across methods (IVW, WME, and MR-Egger) and multiple analyses. The functional relevance and cell-type specificity of these causal biomarkers were examined using published single-cell RNA sequencing data. RESULTS: Proteomic analysis revealed a total of 846 DEPs. MR identified five causal genes in blood (ACOT1, ALDH1A1, DAG1, STEAP4, LIPA) and four causal genes in musculoskeletal tissue (ACOT1, PPIC, TSC22D4, LIPA). ACOT1 and ALDH1A1 exhibited protective effects (β < 0), whereas DAG1, STEAP4, LIPA, PPIC, and TSC22D4 were associated with increased disease risk (β > 0). CONCLUSION: Seven genes (ACOT1, LIPA, PPIC, TSC22D4, ALDH1A1, DAG1, and STEAP4) demonstrating both proteomic dysregulation and genetic causality may serve as novel biomarkers or therapeutic targets for NONFH. These findings provide a foundation for further mechanistic studies and hold potential for advancing diagnosis and treatment strategies for NONFH.