APOBEC mutagenesis is a common process in normal human small intestine

Abstract

APOBEC mutational signatures SBS2 and SBS13 are common in many human cancer types. However, there is an incomplete understanding of its stimulus, when it occurs in the progression from normal to cancer cell and the APOBEC enzymes responsible. Here we whole-genome sequenced 342 microdissected normal epithelial crypts from the small intestines of 39 individuals and found that SBS2/SBS13 mutations were present in 17% of crypts, more frequent than most other normal tissues. Crypts with SBS2/SBS13 often had immediate crypt neighbors without SBS2/SBS13, suggesting that the underlying cause of SBS2/SBS13 is cell-intrinsic. APOBEC mutagenesis occurred in an episodic manner throughout the human lifespan, including in young children. APOBEC1 mRNA levels were very high in the small intestine epithelium, but low in the large intestine epithelium and other tissues. The results suggest that the high levels of SBS2/SBS13 in the small intestine are collateral damage from APOBEC1 fulfilling its physiological function of editing APOB mRNA.

Fig. 1. Burdens and mutational signatures in normal human small intestinal crypts.

a, SBS burden versus age, showing regression lines for the three different sectors of the small intestine. Regression lines were estimated using linear mixed-effects models. Error bands represent 95% confidence interval for the fixed effect of age. Colors indicate biopsy regions, with orange, green and blue representing duodenum, ileum and jejunum, respectively. Shapes indicate whether the donor has a celiac history or not. Crosses indicate donors with a celiac history, and dots indicate donors without a celiac history. b, ID burden versus age, showing regression lines for the three different sectors of the small intestine. c, The proportion of mutations in each crypt attributed to each SBS mutational signature (arranged by ascending age). Signatures are color coded as indicated on the right.

Fig. 2. APOBEC mutagenesis on phylogenetic trees.

Phylogenies of small intestine crypts with mutational signature annotation. Branch lengths correspond to SBS burdens. Signatures exposures are color coded below the trees. a, Phylogeny of PD41851, an individual with frequent APOBEC mutagenesis exhibiting SBS1, SBS5 and SBS18 with SBS2/SBS13 in 8 of 11 crypts. b, Phylogeny of PD41853, exhibiting SBS1, SBS5 and SBS18 with SBS2/SBS13 in 1 of 11 crypts. c, Phylogeny of PD43953, a 4-year-old child exhibiting SBS1, SBS5, SBS18 and SBS2/SBS13. d, Phylogeny of PD41852 with APOBEC mutagenesis detected before a crypt fission event at ~25 years of age. e, Phylogeny of PD43401 with APOBEC mutagenesis detected after a crypt fission event at ~30 years of age. f, Phylogeny of PD43403 with APOBEC mutagenesis detected after a crypt fission event at ~30 years of age.

Fig. 3. Spatial distribution of APOBEC-positive crypts.

APOBEC-positive crypts and their surrounding crypts before microdissection with their SBS mutational spectrums. Signatures exposures are color coded on top left. Red dots, APOBEC-positive crypts. Blue dots, APOBEC-negative crypts that have been sequenced. Gray rectangles on the mutational spectra circle characteristic peaks of SBS2/SBS13. a, PD43401, this individual has one APOBEC-positive crypt but all the remaining crypts in the neighborhood are negative. b, PD52487, with an APOBEC-negative crypt (PD52487b_lo0005) between APOBEC-positive crypts. c, PD45778, an APOBEC-negative crypt (PD45778b_lo0002) between APOBEC-positive crypts.

Fig. 4. Local mutation clusters (kataegis) in the normal small intestine.

a,b, Two examples of crypts with kataegis. Types of SBSs are indicated by the color code below. Red arrowheads show the location of kataegis. a, Rainfall plot of a crypt from PD28690 showing a mutation cluster on chromosome 16. b, Rainfall plot of a crypt from PD28690 showing mutation clusters on chromosomes 7, 14 and 22.

Fig. 5. APOBEC1/APOBEC3A/APOBEC3B expression across normal tissues.

Bulk tissue gene APOBEC expression from The HPA project consensus dataset. Image credit: HPA (v21.proteinatlas.org). a, APOBEC1 bulk tissue gene expression (https://v21.proteinatlas.org/ENSG00000111701-APOBEC1/tissue). Duodenum nTPM = 29, small intestine nTPM = 26 and colon nTPM = 1.1. b, APOBEC3A bulk tissue gene expression (https://v21.proteinatlas.org/ENSG00000128383-APOBEC3A/tissue). Duodenum nTPM = 1.4, small intestine nTPM = 0.8 and colon nTPM = 1.5. c, APOBEC3B (https://v21.proteinatlas.org/ENSG00000179750-APOBEC3B/tissue) bulk tissue gene expression. Duodenum nTPM = 7.3, small intestine nTPM = 4.3 and colon nTPM = 12.1.

Extended Data Fig. 1. Variant allele frequency distributions reflecting clonality of LCM samples.

Variant allele frequency distribution for all individuals in this study. Most crypts have peaks around 0.5, indicating their monoclonity.

Extended Data Fig. 2. Indel spectrum for small intestinal crypts.

Number indicates total number of indels detected. 1 bp deletion and insertion at 6+ T homopolymers are the most common types of Indels.

Extended Data Fig. 3. Phylogenies of all individuals in this study with mutational signature annotation.

Branch lengths correspond to SBS burdens, and color codes for mutational signatures are at the top. Numbers on the tips/branch indicate the number of hypermutation clusters placed on the tips/branch.

Extended Data Fig. 4. Comparison of APOBEC mutagenesis in small bowel cancer and normal crypts.

Boxplot of SBS2/13 exposures in small bowel adenocarcinomas and normal crypts with APOBEC mutagenesis (n = 14 for adenocarcinomas and n = 58 for normal crypts). The central line, box and whiskers represent the median, interquartile range (IQR) from first to third quartiles, and 1.5 × IQR, respectively. Burdens in cancer WES data have been adjusted by the proportion of exomes in genome to compare with whole-genome sequencing data. Median = 1691 (adenocarcinoma) and 242 (normal), two-tailed t-test P = 2 × 10⁻⁴.

Extended Data Fig. 5. Signature burden versus age for SBS1, SBS5, SBS18, SBS2 and SBS13.

Regression lines were estimated using linear mixed models. Error bands represent 95% confidence interval for the fixed effect of age. Colors indicate biopsy regions, with orange, green and blue representing duodenum, ileum and jejunum, respectively. Shapes indicate whether the donor has a celiac history or not. Crosses indicates donors with a celiac history, and dots indicate donors without a celiac history. (a) SBS1 burden versus age, showing regression lines for the three different sectors of the small intestine. (b) SBS5 burden versus age, showing a regression line for all samples because the rate is not statistically different for the three sectors according to linear mixed models. (c) SBS18 burden versus age, showing a regression line for all samples because the rate is not statistically different for the three sectors according to linear mixed models. (d) SBS2 burden versus age, the relationship is not linear. (e) SBS13 burden versus age, the relationship is not linear.

Extended Data Fig. 6. Extended contexts of APOBEC mutations.

Enrichment of pyrimidines (Y) instead of purines (R) at −2 position indicate APOBEC3B is unlikely to be the major contributing enzyme, while APOBEC1/3A could not be excluded. (a) Sequence logo showing the base frequency from −3 to +1 of APOBEC signature from HDP de novo extraction. (b) Fold enrichment of different TC contexts in all phylogenetic branches with SBS2/13, showing a preference for YTCA/TCA/TCW. RTCA: P = 1, YTCA: P = 3.0 × 10⁻⁹⁶, TCA: P = 2.3 × 10⁻³¹, TCW: P = 6.4 × 10⁻⁴, one-tailed Fisher’s exact test.

Extended Data Fig. 7. Comparisons of the effects of age and celiac disease on substitutions and Indels.

Central dots represent the estimated fixed effect from linear mixed-effects models, and error bars represent the 95% confidence intervals. N = 306 crypts. (a) Age and celiac disease effect on different single-base substitution mutational signatures. (b) Age and celiac disease effect on Indels.