www.nature.com/scientificreports OPEN A search for modifying genetic factors in CHEK2:c.1100delC breast cancer patients Camilla Wendt1*, Taru A. Muranen2, Lotta Mielikäinen2, Jessada Thutkawkorapin3, Carl Blomqvist4, Xiang Jiao3, Hans Ehrencrona5, Emma Tham3, Brita Arver6, Beatrice Melin7, Ekaterina Kuchinskaya8, Marie Stenmark Askmalm8, Ylva Paulsson‑Karlsson9, Zakaria Einbeigi10, Anna von Wachenfeldt Väppling1, Eija Kalso11, Tiina Tasmuth11, Anne Kallioniemi12, Kristiina Aittomäki13, Heli Nevanlinna2, Åke Borg14 & Annika Lindblom3 The risk of breast cancer associated with CHEK2:c.1100delC is 2–threefold but higher in carriers with a family history of breast cancer than without, suggesting that other genetic loci in combination with CHEK2:c.1100delC confer an increased risk in a polygenic model. Part of the excess familial risk has been associated with common low‑penetrance variants. This study aimed to identify genetic loci that modify CHEK2:c.1100delC‑associated breast cancer risk by searching for candidate risk alleles that are overrepresented in CHEK2:c.1100delC carriers with breast cancer compared with controls. We performed whole‑exome sequencing in 28 breast cancer cases with germline CHEK2:c.1100delC, 28 familial breast cancer cases and 70 controls. Candidate alleles were selected for validation in larger cohorts. One recessive synonymous variant, rs16897117, was suggested, but no overrepresentation of homozygous CHEK2:c.1100delC carriers was found in the following validation. Furthermore, 11 non‑synonymous candidate alleles were suggested for further testing, but no signifcant diference in allele frequency could be detected in the validation in CHEK2:c.1100delC cases compared with familial breast cancer, sporadic breast cancer and controls. With this method, we found no support for a CHEK2:c.1100delC‑specifc genetic modifer. Further studies of CHEK2:c.1100delC genetic modifers are warranted to improve risk assessment in clinical practice. Breast cancer aggregates in families and has a considerable inherited component. Approximately 20% of the genetic risk for breast cancer is explained by pathogenic mutations in the high-penetrance genes BRCA1, BRCA2, TP53, STK11 and PTEN1. Other rare, intermediate-risk variants, such as PALB2, CHEK2 and ATM account for about 5% of the inherited risk 2,3 and common low-risk variants for another 18–19%4–6. Checkpoint kinase 2 is a protein product of the CHEK2 gene that localizes to chromosome 22q12.1. It is part of the network that responds to DNA damage in order to maintain genomic integrity7. Te protein-truncating variant CHEK2:c.1100delC is associated with a two-threefold risk of breast cancer 8,9. In women with familial aggregation of breast cancer, the risk is even higher. An odds ratio of up to 4.8 has been seen in women with a family history of breast cancer, which is equivalent to a 37% cumulative risk of breast cancer by the age of 1Department of Clinical Science and Education, Karolinska Institutet, Södersjukhuset, Stockholm, Sweden. 2Department of Obstetrics and Gynecology, Helsinki University Hospital, University of Helsinki, Helsinki, Finland. 3Department of Molecular Medicine and Surgery, Karolinska Institutet, Solna, Stockholm, Sweden. 4Department of Oncology, Helsinki University Hospital, University of Helsinki, Helsinki, Finland. 5Department of Clinical Genetics and Pathology, Ofce for Medical Services, Region Skåne, Lund, Sweden. 6Department of Oncology-Pathology, Karolinska Institutet, Solna, Stockholm, Sweden. 7Department of Radiation Sciences, Oncology, Umeå University, Umeå, Sweden. 8Department of Clinical Genetics, Department of Clinical Experimental Medicine, Linköping University, Linköping, Sweden. 9Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. 10Department of Oncology, Sahlgrenska University Hospital, 41345 Göteborg, Sweden. 11Department of Anaesthesiology, Intensive Care, and Pain Medicine, Helsinki University Hospital and University of Helsinki, Helsinki, Finland. 12TAYS Cancer Centre and Faculty of Medicine and Health Technology, Tampere University; Fimlab Laboratories, Tampere University Hospital, Tampere, Finland. 13Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland. 14Department of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Medicon Village, Lund, Sweden. *email: [email protected] Scientifc Reports | (2021) 11:14763 | https://doi.org/10.1038/s41598-021-93926-x 1 Vol.:(0123456789) www.nature.com/scientificreports/ 70 years8–10. In addition, the c.1100delC allele has been associated with younger age at onset, a threefold increased risk of a second breast cancer, as well as a worse prognosis among women with oestrogen receptor-positive cancer9,11,12. Te considerably higher risk in women with a family history of breast cancer is in accordance with the sug- gested polygenic model where several susceptibility loci together confer a multiplicative efect on breast cancer risk13,14. Te fact that the model also can be applied to CHEK2:c.1100delC carriers is supported by a study of low-risk breast cancer variants in 34 000 women with and without a family history of breast cancer. A polygenic risk score (PRS) that was based on the combined risk of 74 low risk variants was calculated. Te result suggested that the polygenic risk score could be used to stratify risk in c.1100delC carriers and that the low-risk variants explained a part of the familial risk. Te authors estimated that 20% of CHEK2:c.1100delC carriers with the highest PRS had an estimated lifetime breast cancer risk of > 30%. Correspondingly, 20% of carriers with the lowest PRS had an estimated lifetime risk of 14% which is close to the average population risk 15. A synergistic efect between low-risk variants and BRCA1 and BRCA2 mutations has also been shown16. Te risk for muta- tion carriers being afected is thus modifed by other genetic variants and family history in addition to lifestyle factors. A risk prediction model, the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) has been developed to calculate the lifetime risk of breast cancer, including carriers of a moderate-penetrance allele such as CHEK2:c.1100delC. Te BOADICEA model allows risk stratifcation for established genetic and non-genetic risk factors17. Still, other causative gene variants possibly remain to be identifed, since the previously identifed low-, intermediate-, and high-risk genes cover less than half of the estimated heritable component. Characterising factors that increase the risk in carriers of moderate-risk alleles is important, in order to identify the high-risk group that benefts most from preventive interventions. In this study, we used whole-exome sequencing of a CHEK2:c.1100delC positive cohort with familial breast cancer, to identify putative risk modifying alleles. In the frst phase we aimed to fnd candidate risk alleles for further validation in the second phase with larger cohorts of CHEK2:c.1100delC positive cases and controls. Results We performed whole-exome sequencing in 28 breast cancer cases with germline CHEK2:c.1100delC, 28 familial breast cancer cases and 70 controls. Candidate alleles were selected for validation in larger cohorts (Fig. 1). Recessive variants. We analysed the exome sequencing data for a discovery of rare homozygous variants in CHEK2:c.1100C carriers, to identify risk alleles with recessive inheritance pattern. Only one variant was sug- gested, rs16897117. Among the 28 CHEK2 carriers, there were 3 patients homozygous for rs16897117, whereas among the non-carrier breast cancer cases or healthy controls, there were no rs16897117 homozygotes. We set up to test the hypothesis of rs1689711 being a CHEK2:c.1100C risk modifer in larger sample collections, starting with 67 CHEK2 patients, as well as 688 non-carrier breast cancer cases and 246 healthy controls. Tis study confrmed the skewed allele distribution, with fewer individuals heterozygous for rs16897117 among the CHEK2 patients than among non-carrier patients or healthy controls. In a case-only analysis, the odds ratio between rs16897117 rare allele (A) and CHEK2:c.1100delC was 0.46 (95% confdence interval CI 0.17–1.04, P 0.053 (Table 1: SWEA1). Next, we did another follow-up using 45 CHEK2 carriers plus 87 familial breast cancer patients and 47 con- trols from the Swedish cohorts. None of the CHEK2 carriers or the familial breast cancer patients were found to be homozygous for the rs16897117 variant. Te only two homozygous individuals of this follow-up were identifed in the control group. No skewness in allele distribution was observed in any of these groups (Table 1: SWEA2). Te results seemed less clear, but to resolve this, we tested the association between rs16897117 and c.1100delC in a Finnish population, where the c.1100delC allele has a relatively high, 1.2%, frequency18. Geno- typing of three independent patient series identifed a single c.1100delC carrier patient, who was homozygous for rs16897117. Te skewed allele distribution for rs16897117 was observed in the Helsinki cohorts, but not in the Tampere cohort. A study-stratifed OR for association between rs16897117 and c.1100delC, combining all cohorts from Sweden and Finland, was 0.69 (95% CI 0.46–1.03, P 0.073), encouraging further analysis. Finally, the genotype data for rs16897117 and c.1100delC were obtained from the OncoArray project of the Breast Cancer Association Consortium5. Te availability of a good number