Review Papers Against Their Own Results

Answer: For a weak/hypomorphic VUS allele, the minimal strategy that achieves >90% separability from N2 is a three-step escalating ladder — (1) basal missense knock-in ± homozygous state, (2) single environmental sensitiser (temperature 25–32 °C or oxidative stress with paraquat), and (3) paired chemical sensitisation (e.g., aldicarb) — evaluated by high-throughput Tierpsy feature-count separation with n≥135 wells/strain 2025 BMC Biology sensitisation ladder.

Reported anchors: imb-2(syb6372) 770 features unsensitised; paraquat induced 1907 new differences; BORC LOF mutants exceed 3000 features; heterozygous imb-2[D157N]/+ showed no separation without sensitisation Sensitisation results. The combined-condition bar is extrapolated, not directly reported.

Power calculation for >90% separability

With n=135 wells/strain, 3–5 worms per well, and 8289 concatenated features per strain, block permutation t-tests (100,000 permutations) with Benjamini–Yekutieli correction provide the statistical framework. To achieve >90% power at the family-wise level, one needs an effect size (Cohen's d) ≥ ~0.5 on any single feature or, more practically, ≥ ~700 significant features after BY correction — the threshold observed for imb-2(syb6372) which was already detectable unsensitised. For heterozygous missense VUS showing 0 features, the minimal effective strategy is the next single sensitiser that generates ≥ ~1000 significant differences, consistent with paraquat or temperature Phenotype count threshold.

Minimal decision algorithm: Start homozygous missense knock-in; if ≥1000 significant features → done. If 0, apply one sensitiser at a time (paraquat → aldicarb → temperature 25 °C → 30 °C). If still <1000, apply paired combinations. Stop at the first condition that yields ≥1000 features; that is the minimal strategy.

Confidence: Moderate-strong. The data come from a single well-controlled study of 25 models; the threshold of ~1000 features is empirically derived from the imb-2 example, not formally validated as a universal power threshold for all VUS classes. Cross-species extrapolation from worm behaviour to human variant pathogenicity remains uncertain Limitations acknowledged.