Evaluation of the association of heterozygous germline variants in NTHL1 with breast cancer predisposition: an international multi-center study of 47,180 subjects

Abstract

Bi-allelic loss-of-function (LoF) variants in the base excision repair (BER) gene NTHL1 cause a high-risk hereditary multi-tumor syndrome that includes breast cancer, but the contribution of heterozygous variants to hereditary breast cancer is unknown. An analysis of 4985 women with breast cancer, enriched for familial features, and 4786 cancer-free women revealed significant enrichment for NTHL1 LoF variants. Immunohistochemistry confirmed reduced NTHL1 expression in tumors from heterozygous carriers but the NTHL1 bi-allelic loss characteristic mutational signature (SBS 30) was not present. The analysis was extended to 27,421 breast cancer cases and 19,759 controls from 10 international studies revealing 138 cases and 93 controls with a heterozygous LoF variant (OR 1.06, 95% CI: 0.82-1.39) and 316 cases and 179 controls with a missense variant (OR 1.31, 95% CI: 1.09-1.57). Missense variants selected for deleterious features by a number of in silico bioinformatic prediction tools or located within the endonuclease III functional domain showed a stronger association with breast cancer. Somatic sequencing of breast cancers from carriers indicated that the risk associated with NTHL1 appears to operate through haploinsufficiency, consistent with other described low-penetrance breast cancer genes. Data from this very large international multicenter study suggests that heterozygous pathogenic germline coding variants in NTHL1 may be associated with low- to moderate- increased risk of breast cancer.

Fig. 1. Genomic characterization of breast and ovarian cancers from carriers of NTHL1 germline loss-of-function variants.

a Germline variants, somatic mutations and HRD score of NTHL1 associated tumors. Germline and somatic variant types are color-coded according to the legend. The phenobar provides information about estrogen receptor (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2) status, and somatic mutations in driver genes. b The weighted contribution of mutational signatures from breast cancers of NTHL1 germline variant carriers. c Fraction of genome altered (FGA) and d Homologous recombination deficiency score (HRD) for NTHL1-null (n = 1) and NTHL1-het (n = 14) breast cancers compared to breast cancers from PALB2 (n = 15) and RAD51C (n = 9) germline LoF variant carriers, and sporadic breast cancers (n = 115).

Fig. 2. NTHL1 protein expression in sporadic breast cancer (n = 21) and NTHL1-het breast cancer (n = 8).

a NTHL1 expression in sporadic breast cancer and NTHL1-het cancer of ER+, HER2+, and triple-negative types. Multiplex immunofluorescent staining approach was used and the fluorescence signal was displayed in colorimetric pattern for better contrast. NTHL1: brown color; DIPI: blue color. The epithelial marker AE1/AE3 (Supplementary Fig. 4) was used to identify cancer cells in the breast cancer tissue in NTHL1 expression quantitation in (b) and (c). Scale bar = 100 μm. b The average intensity of NTHL1 in sporadic cancer group compared to NTHL1-het group. c The average intensity of NTHL1 in sporadic cancer group compared to NTHL1-het group according to ER, PR, and HER2 status. BC breast cancer, TNBC triple-negative breast cancer.

Fig. 3. Frequency of heterozygous germline variants in NTHL1 and odds ratios for breast cancer observed in case–control data from ten multicenter international cohorts.

a Heterozygous loss-of-function variants in NTHL1 and odds ratios in ten case–control cohorts. b Heterozygous missense variants in NTHL1 and odds ratios in ten case–control cohorts. The overall effect of odds ratios were computed based on a fixed-effect model. BEACCON hereditary BrEAst Case CONtrol study, SEARCH UK Population-based Breast Cancer Study, GC-HBOC German Consortium for Hereditary Breast and Ovarian Cancer, GENESIS French familial BRCAx study (some data were previously published), VHIO familial breast cancer and control study of the Vall d’Hebron Institute of Oncology of Barcelona, OFBCR Ontario Familial Breast Cancer Registry, DFBBCS the Dutch Familial Bilateral Breast Cancer Study, HABC Hispanic-American Breast Cancer Study, ABCFR Australian Breast Cancer Family Registry, CARTaGENE Québec Population-based Breast Cancer Study.

Fig. 4. Germline NTHL1 variants identified in 27,421 cases and 19,759 controls.

a Lollipop plot of all LoF variants identified in 27,421 cases and 19,759 controls. b Odds ratio (OR) and 95% confidence interval (95% CI) for recurrent LoF variants p.(Gln90Ter) and all the rest of rare LoF variants (MAF < 0.001 according to gnomAD). c Lollipop plot of all missense variants identified in 27,421 cases and 19,759 controls. Nth, Endonuclease III. d OR and 95% CI for missense variants located in functional domain and the rest of the NTHL1 protein.