Genetically Predicted Thyroid-Stimulating Hormone and Coronary Artery Disease: Lipid-Mediated Causal Pathways, PCSK9 Protein Validation, and a Suppression Effect Revealed by Multi-Layered Mendelian Randomization

Background Subclinical hypothyroidism (SCH) affects 4–10% of the general population and has been inconsistently linked to coronary artery disease (CAD). Whether thyroid-stimulating hormone (TSH) causally influences CAD risk, and through which biological pathways, remains unresolved. We conducted a multi-layered Mendelian randomization (MR) study integrating mediation analysis, proteomics-based drug-target MR, and genetic colocalization to dissect the causal architecture of the TSH–CAD relationship. Methods Two-sample MR was performed using 63 genome-wide significant instruments for TSH (Zhou et al., HUNT/ThyroidOmics, N = 119,715) against CAD (CARDIoGRAMplusC4D, 60,801 cases/123,504 controls). Two-step mediation MR with Sobel test evaluated five candidate pathways: metabolic (LDL-C, triglycerides, HDL-C), inflammatory (CRP), and coagulation (fibrinogen). Drug-target MR used five independent PCSK9 cis-pQTL instruments from deCODE proteomics (N = 35,363). Bayesian colocalization was performed at the PCSK9 locus. Sensitivity analyses included IVW (fixed and random effects), weighted median, MR-Egger, MR-PRESSO, Steiger directionality test, and reverse MR (CAD→TSH). Results Genetically predicted TSH showed no overall association with CAD (IVW: OR = 0.987, 95% CI: 0.943–1.032, P = 0.557; 62 SNPs). However, TSH was robustly associated with increased LDL-C (β = 0.077, P = 1.4×10⁻⁵; confirmed by weighted median P = 0.043 and MR-PRESSO corrected P = 7.4×10⁻⁴), with no evidence of pleiotropy (Egger intercept P = 0.983). The triglyceride association was borderline (IVW P = 0.053; MR-PRESSO corrected P = 0.010). No robust associations were found with HDL-C (P = 0.291) or CRP (IVW P = 0.039; MR-PRESSO corrected P = 0.838 after removing 6 outliers). Two-step mediation MR identified LDL-C as the sole significant mediator (indirect effect β = 0.029, Sobel P < 0.001); the triglyceride pathway was borderline (Sobel P = 0.059). A suppression effect was identified: the harmful LDL-mediated indirect effect (+ 0.029) was offset by a protective direct effect (− 0.043), yielding the observed null total effect (suppression ratio = − 2.1). Drug-target MR confirmed that genetically proxied PCSK9 protein levels causally increased CAD risk (IVW: OR = 1.210, P = 5.3×10⁻⁷; weighted median P = 5.2×10⁻⁴; Egger intercept P = 0.923). Reverse MR confirmed no evidence of reverse causality (CAD→TSH: β=−0.003, P = 0.825). Strong colocalization was observed between LDL-C and CAD at the PCSK9 locus (PPH4 = 0.915), but not for triglycerides (PPH4 = 0.251) or CRP (PPH4 = 0.002). Conclusions TSH influences CAD risk predominantly through an LDL-C–mediated pathway involving the TSHR–SREBP2–PCSK9–LDLR axis, as validated by convergent evidence from mediation MR, proteomics-based MR, and colocalization. The null total effect reflects a suppression phenomenon where harmful lipid-mediated and protective lipid-independent effects of TSH counterbalance each other. These findings support prioritizing lipid-lowering therapy—particularly PCSK9-targeted interventions—over TSH normalization alone for cardiovascular risk management in SCH.