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. 2023 Jan 17;29(2):422-431.
doi: 10.1158/1078-0432.CCR-22-1703.

NBN Pathogenic Germline Variants are Associated with Pan-Cancer Susceptibility and In Vitro DNA Damage Response Defects

Sami Belhadj  1   2   3 , Aliya Khurram  1 , Chaitanya Bandlamudi  4 , Guillermo Palou-Márquez  5 , Vignesh Ravichandran  4 , Zoe Steinsnyder  1 , Temima Wildman  1 , Amanda Catchings  1 , Yelena Kemel  6 , Semanti Mukherjee  1 , Benjamin Fesko  6 , Kanika Arora  4 , Miika Mehine  4 , Sita Dandiker  1 , Aalin Izhar  1 , John Petrini  6 , Susan Domchek  7 , Katherine L Nathanson  7 , Jamie Brower  7 , Fergus Couch  8 , Zsofia Stadler  1 , Mark Robson  1   9 , Michael Walsh  1   10 , Joseph Vijai  1   11 , Michael Berger  4 , Fran Supek  5   12 , Rachid Karam  3 , Sabine Topka  1   2 , Kenneth Offit  1   2   6   11
Affiliations

NBN Pathogenic Germline Variants are Associated with Pan-Cancer Susceptibility and In Vitro DNA Damage Response Defects

Sami Belhadj et al. Clin Cancer Res. .

Abstract

Purpose: To explore the role of NBN as a pan-cancer susceptibility gene.

Experimental design: Matched germline and somatic DNA samples from 34,046 patients were sequenced using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets and presumed pathogenic germline variants (PGV) identified. Allele-specific and gene-centered analysis of enrichment was conducted and a validation cohort of 26,407 pan-cancer patients was analyzed. Functional studies utilized cellular models with analysis of protein expression, MRN complex formation/localization, and viability assessment following treatment with γ-irradiation.

Results: We identified 83 carriers of 32 NBN PGVs (0.25% of the studied series), 40% of which (33/83) carried the Slavic founder p.K219fs. The frequency of PGVs varied across cancer types. Patients harboring NBN PGVs demonstrated increased loss of the wild-type allele in their tumors [OR = 2.7; confidence interval (CI): 1.4-5.5; P = 0.0024; pan-cancer], including lung and pancreatic tumors compared with breast and colorectal cancers. p.K219fs was enriched across all tumor types (OR = 2.22; CI: 1.3-3.6; P = 0.0018). Gene-centered analysis revealed enrichment of PGVs in cases compared with controls in the European population (OR = 1.9; CI: 1.3-2.7; P = 0.0004), a finding confirmed in the replication cohort (OR = 1.8; CI: 1.2-2.6; P = 0.003). Two novel truncating variants, p.L19* and p.N71fs, produced a 45 kDa fragment generated by alternative translation initiation that maintained binding to MRE11. Cells expressing these fragments showed higher sensitivity to γ-irradiation and lower levels of radiation-induced KAP1 phosphorylation.

Conclusions: Burden analyses, biallelic inactivation, and functional evidence support the role of NBN as contributing to a broad cancer spectrum. Further studies in large pan-cancer series and the assessment of epistatic and environmental interactions are warranted to further define these associations.

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Figures

Figure 1. Germline NBN variation in >34,000 patients. A, Distribution and frequency of identified NBN PGVs. Protein domains are represented as reported previously (54). Golden arrows indicate variants that are reported as P/LP in ClinVar (Jan 2021) with more than two gold stars. FHA: forkhead-associated domain, BRCT: breast cancer C-terminal domains, MRE11: MRE11 interaction region, ATM: ATM-binding domain. B, Positivity rate of PGVs shows notable differences between cancer types. Only cancer types diagnosed in >500 patients are represented. The complete data are available in Supplementary Table S1. C, Multicarriers of NBN PGVs in addition to a P/LP variant in a cancer susceptibility gene reported in ClinVAR. D, Somatic loss of the WT allele is significantly more frequent in P/LP variants compared with B/LB ones, as classified by PathoMAN (top), as well as in truncating variants when compared with silent ones (bottom). WT-loss events appear to be slightly more enriched in missense compared silent germline NBN variants. WT-Loss: somatic loss of the WT allele, VAR-Loss: somatic loss of the variant (mutated) allele.
Figure 1.
Germline NBN variation in >34,000 patients. A, Distribution and frequency of identified NBN PGVs. Protein domains are represented as reported previously (54). Golden arrows indicate variants that are reported as P/LP in ClinVar (Jan 2021) with more than two gold stars. ATM, ATM-binding domain; BCRT, breast cancer C-terminal domains; FHA, forkhead-associated domain; MRE11, MRE11 interaction region. B, Positivity rate of PGVs shows notable differences between cancer types. Only cancer types diagnosed in >500 patients are represented. The complete data are available in Supplementary Table S1. C, Multicarriers of NBN PGVs in addition to a P/LP variant in a cancer susceptibility gene reported in ClinVAR. D, Somatic loss of the WT allele is significantly more frequent in P/LP variants compared with B/LB ones, as classified by PathoMAN (top), as well as in truncating variants when compared with silent ones (bottom). WT-loss events appear to be slightly more enriched in missense compared silent germline NBN variants. VAR-Loss, somatic loss of the variant (mutated) allele; WT-Loss, somatic loss of the WT allele.
Figure 2. Cosegregation study in families with NBN PGVs identified in >34,000 patients (MSKCC) as well as families identified through the PROMPT. Carrier status is indicated in blue: noncarriers (−/−), heterozygous (±), and homozygous (+/+). Filled black symbol: affected with cancer. Black arrow: index case. Ages at diagnosis are provided after cancer type. Ages at information gathering or at death are provided in the top right corner.
Figure 2.
A–E, Cosegregation study in families with NBN PGVs identified in >34,000 patients (MSKCC) as well as families identified through the PROMPT. Carrier status is indicated in blue: noncarriers (−/−), heterozygous (+/−), and homozygous (+/+). Filled black symbol: affected with cancer. Black arrow: index case. Ages at diagnosis are provided after cancer type. Ages at information gathering or at death are provided in the top right corner.
Figure 3. Functional characterization of NBN p.L19* and p.N71fs in NBS-ILB1 cell line. A, qPCR Validation of the overexpression of mutant cDNAs. B, Western blots revealed the generation of a novel truncated protein fragment of ∼45 kDa (p45). C, C-terminal HA-tagging confirmed the binding of p45 to MRE11. D, Identification of p.M389 as the preferential alternative translation initiation site for the expression of p45. E, γ-irradiation exposure shows impaired ability of p.L19* and p.N71fs to rescue cell viability, similar to p.K219fs models. Significance was assessed by two-way ANOVA (*, P ≤ 0.05; ****, P ≤ 0.0001). F, Activation of the ATM axis as measured by KAP1 phosphorylation is impaired in p.L19* and p.N71fs following γ-irradiation.
Figure 3.
Functional characterization of NBN p.L19* and p.N71fs in NBS-ILB1 cell line. A, qPCR Validation of the overexpression of mutant cDNAs. B, Western blots revealed the generation of a novel truncated protein fragment of ∼45 kDa (p45). C, C-terminal HA-tagging confirmed the binding of p45 to MRE11. D, Identification of p.M389 as the preferential alternative translation initiation site for the expression of p45. E, γ-irradiation exposure shows impaired ability of p.L19* and p.N71fs to rescue cell viability, similar to p.K219fs models. Significance was assessed by two-way ANOVA (*, P ≤ 0.05; ****, P ≤ 0.0001). F, Activation of the ATM axis as measured by KAP1 phosphorylation is impaired in p.L19* and p.N71fs following γ-irradiation.
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