# Functional & Integrative Genomics revision: locked analysis plan

## Scope and evidence boundary

This revision treats Yangxin Decoction as a case study for candidate nomination, not as a proven therapy. The confirmatory question is whether a prespecified seven-gene PI3K-AKT panel (AKT1, PIK3CA, PIK3R1, GSK3B, MTOR, PTEN, STAT3) receives convergent support from cardiac/blood cis-eQTL genetics and human cardiac transcriptomics. PSEN2 and ITGB3 are retained as negative-control genes. Existing colocalization results generated from the eQTLGen FDR-filtered file are excluded from inferential use because colocalization requires complete local association statistics.

## Genetic analysis

- Exposure resource: eQTL Catalogue/GTEx v8 uniformly processed gene-expression QTL summary statistics (study QTS000015).
- Tissues: heart left ventricle (QTD000256, n=382) and whole blood (QTD000356, n=670).
- Cis region: every tested variant reported for the gene in the QTL Catalogue file, queried over gene coordinates plus/minus 1 Mb on GRCh38.
- Outcomes: FinnGen R12 I9_CARDMYO (7,649 cases; 375,343 controls), I9_MYOCARD (2,062 cases; 252,810 controls), and I9_HEARTFAIL (37,653 cases; 462,695 controls), using the official R12 manifest.
- Instrument rule: the strongest genome-wide-significant cis-eQTL (P < 5e-8, MAF >= 0.01, F=(beta/SE)^2 > 10) is selected independently of outcome. A single-SNP Wald ratio is reported as exploratory. Genes without a qualifying instrument are reported as not instrumentable; no relaxed P-value threshold is used.
- Multiplicity: Benjamini-Hochberg FDR and Bonferroni correction are applied across all primary gene-by-tissue-by-outcome MR tests actually performed. Negative-control tests form a separate family.
- Colocalization: coloc.abf with p1=1e-4, p2=1e-4, p12=1e-5; p12 sensitivity at 1e-6 and 1e-4. Inputs use complete local GTEx statistics and outcome beta/SE, with tissue-specific MAF and correct case fractions. PP.H4 >= 0.80 is treated as strong support, 0.50-0.80 as moderate, and lower values as not supporting colocalization. The single-causal-variant assumption is retained as an explicit limitation because LD matrices for coloc.susie are not available for every locus.

## Transcriptomic analysis

- Bulk layer: all 61 Tier-1 genes across the four existing GEO analyses. Dataset-level limma BH-adjusted P values are retained; cross-dataset evidence is summarized without converting proxy phenotypes into DCM- or viral-myocarditis-specific claims.
- Single-nucleus layer: the existing donor-level pseudobulk DESeq2 analysis is retained for the eight prespecified hub/pathway genes because the original disease h5ad is not stored locally. The 61-gene cell-type expression matrices are used for localization only, not for disease differential-expression claims.
- Any discrepancy between bulk and single-nucleus results is described as cell-type-specific regulation masked by tissue averaging; cardiomyocyte loss is not inferred because the deconvolution calibration failed.

## Molecular docking

- Archived PDBQT receptor and ligand inputs are rerun with AutoDock Vina 1.2.7.
- Fixed search boxes are those archived in each target config; scoring=vina, exhaustiveness=32, num_modes=20, energy_range=3, min_rmsd=1.0 A, spacing=0.375 A.
- Three prespecified seeds are used per ligand-target pair to assess stochastic reproducibility.
- Docking is descriptive only. It is not called binding validation, target engagement, or therapeutic evidence because archived receptor/ligand preparation provenance is incomplete and a matched decoy set is unavailable.
- Molecular dynamics is not run locally because GROMACS is absent and the current machine has 16 GB RAM and no configured GPU workflow. No MD result will be stated or implied.

## Reporting gate

Claims may only advance when supported by the regenerated tables. Null and unavailable results remain in the main evidence matrix. Figures are delivered only as PDF and undergo page rendering/visual inspection. The manuscript DOCX is created in a separate Functional & Integrative Genomics submission directory and rendered page-by-page before delivery.
