Paired tumor-normal WGS with LOH analysis at POLE locus

[bloo-berries]

What it tests

Whether the wild-type POLE allele has been somatically lost in tumor tissue (Knudson two-hit model), which would explain ultra-hypermutation through complete loss of POLE proofreading rather than dominant-negative gain of function.

Method

• Paired tumor-normal whole-genome sequencing (≥60x tumor, ≥30x normal)
• Allele-specific copy number analysis at the POLE locus (12q24.33) using ASCAT and/or FACETS
• Assess for deletion, copy-neutral LOH (mitotic recombination), or promoter methylation of the wild-type allele

Expected outcomes by model

• Model 1 (LOH) — supported if: Loss of wild-type allele detected in tumor with retention of c.138del allele
• Model 2 (Reinitiation) — neutral: LOH status does not directly confirm or exclude reinitiation
• Model 3 (Poisoning) — weakened if: LOH present (holoenzyme poisoning would not require second hit)
• Model 4 (Haploinsufficiency) — partially weakened if: LOH fully explains tumor phenotype (but cannot explain congenital IVC anomaly or multi-system non-neoplastic findings)

Priority justification

Tier-1: Achievable with existing banked tumor tissue and standard WGS pipelines. LOH is the single most likely explanation for tumor-specific ultra-hypermutation and must be assessed before downstream mechanistic experiments. Directly impacts variant classification (tumor suppressor vs. dominant-negative paradigm).

Key references

• Knudson AG (1971) Mutation and cancer: statistical study of retinoblastoma. PNAS 68:820–823.
• Yoshida M et al. (2022) Frequent somatic gene conversion as a mechanism for LOH. Genome Research.
• See analysis/loh_analysis/README.md for pipeline specifications.