Ultra-deep duplex sequencing reveals unique features of somatic evolution in the normal tissues of a family with Li-Fraumeni syndrome

Key results

1. SuggestedGermline p.R181H was associated with higher mutation frequency (MF) in a neutral 20-region mutagenesis panel. Coding MF in clonal hematopoiesis (CHIP) genes tracked age and chemotherapy but not carrier status, and CHIP clone burden tracked age only.

   Multivariate Gaussian generalized linear models (covariates: age, LFS, chemotherapy) on blood at mean duplex depth 15,683× (12,606× - 18,282×). Pooling all individuals, tissues and panels, the study found 1,992 CHIP coding and 6,487 mutagenesis-panel mutations. Mutagenesis MF showed no chemotherapy effect. TP53 non-coding MF was linked to p.R181H; TP53 coding MF tracked only chemotherapy. · Ultra-deep sequencing of a family carrying…; Germline p.R181H does not affect most CHIP…; Chemotherapy increases TP53 clonal expansions in…; Regression analysis

2. SuggestedSelection on CHIP genes was mostly independent of carrier status (positive for DNMT3A and TET2, negative for GATA2). TP53 was the exception: it lacked positive selection in carriers, and untreated carriers had TP53 dN/dS below 1 once chemotherapy-exposed people were analyzed separately.

   TP53 was the only gene whose bootstrapped (5,000 resamples) dN/dS interquartile ranges did not overlap between groups. In untreated carriers LFS02/LFS03, 4/7 (57%) TP53 coding mutations were non-synonymous vs 18/22 (82%) in non-LFS individuals (not significant). Chemotherapy raised TP53 dN/dS in both groups. · Germline p.R181H does not affect most CHIP…; Chemotherapy increases TP53 clonal expansions in…; Testing for selection

3. ShownAcross the proband's autopsy samples, TP53 coding MF was elevated in sun-exposed skin, blood, normal esophagus and both chronically inflamed gastric samples. All tumors had lost the wild-type TP53 allele and showed no excess coding MF.

   Sun-exposed skin had more than twice the TP53 mutations of the next sample, many of them UV-type CC>TT dinucleotides (SBS7a/b/d). Primary and metastatic samples had p.R181H variant allele frequency >0.5. SBS17a/b appeared in esophageal tumors, metastases and one normal esophagus. Four samples at 2,118-7,376× depth were excluded from MF. · Ultra-deep sequencing of a family carrying…; Mutation frequency and signatures vary across…; Mutational spectrum and signature analysis

4. ShownA TP53 codon 248 substitution (p.R248Q or p.R248W, both inactivating and dominant negative) recurred at low frequency in most of the proband's samples. TP53 mutations were enriched in the DNA-binding domain and had high AlphaMissense scores, consistent with convergent selection.

   18/28 tissue samples carried a codon 248 mutation, and 9/19 were seen in >1 duplex read. The largest buffy-coat clone had frequency 0.0005, and most blood mutations were not shared across tissues, arguing against blood carryover. In cohort blood, 75% of TP53 SNVs fell in the DNA-binding domain vs 46% expected (binomial test). · Chemotherapy increases TP53 clonal expansions in…; TP53 R248Q/W is a mutational hotspot recurrently…

5. ShownAmong somatic TP53 mutations that could be phased against the germline site, most arose on the chromosome copy without p.R181H, pointing to selection for second hits on the wild-type allele.

   Duplex reads spanning the germline site (hg38 chr17:7675070; candidate variants within 150 bp) phased 29 mutations across LFS01, LFS02 and LFS03. Of these, 25/29 lay on the non-carrier copy (binomial test, 0.0001), and all spanning reads agreed on phase. · Allelic specificity but not tissue-context…; Testing for selection