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Table.  Frequencies of Heterozygous Loss-of-Function Germline Mutations Within the FANCM Gene in Cases and Controls
Frequencies of Heterozygous Loss-of-Function Germline Mutations Within the FANCM Gene in Cases and Controls
1.
Easton  DF, Pharoah  PD, Antoniou  AC,  et al.  Gene-panel sequencing and the prediction of breast-cancer risk.  N Engl J Med. 2015;372(23):2243-2257.PubMedGoogle ScholarCrossref
2.
Meindl  A, Hellebrand  H, Wiek  C,  et al.  Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as a human cancer susceptibility gene.  Nat Genet. 2010;42(5):410-414.PubMedGoogle ScholarCrossref
3.
Antoniou  AC, Casadei  S, Heikkinen  T,  et al.  Breast-cancer risk in families with mutations in PALB2 N Engl J Med. 2014;371(6):497-506.PubMedGoogle ScholarCrossref
4.
Ciccia  A, Ling  C, Coulthard  R,  et al.  Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM.  Mol Cell. 2007;25(3):331-343.PubMedGoogle ScholarCrossref
5.
Gracia-Aznarez  FJ, Fernandez  V, Pita  G,  et al.  Whole exome sequencing suggests much of non-BRCA1/BRCA2 familial breast cancer is due to moderate and low penetrance susceptibility alleles.  PLoS One. 2013;8(2):e55681.PubMedGoogle ScholarCrossref
6.
Kiiski  JI, Pelttari  LM, Khan  S,  et al.  Exome sequencing identifies FANCM as a susceptibility gene for triple-negative breast cancer.  Proc Natl Acad Sci U S A. 2014;111(42):15172-15177.PubMedGoogle ScholarCrossref
7.
Peterlongo  P, Catucci  I, Colombo  M,  et al.  FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor.  Hum Mol Genet. 2015;24(18):5345-5355.PubMedGoogle ScholarCrossref
8.
Cybulski  C, Wokołorczyk  D, Jakubowska  A,  et al.  Risk of breast cancer in women with a CHEK2 mutation with and without a family history of breast cancer.  J Clin Oncol. 2011;29(28):3747-3752.PubMedGoogle ScholarCrossref
Brief Report
September 2017

Association Between Loss-of-Function Mutations Within the FANCM Gene and Early-Onset Familial Breast Cancer

Author Affiliations
  • 1Center for Familial Breast and Ovarian Cancer and Center for Integrated Oncology, Medical Faculty, University Hospital Cologne, Cologne, Germany
  • 2Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
  • 3Department of Gynaecology and Obstetrics, Klinikum rechts der Isar der Technischen Universität München, Munich, Germany
  • 4Department of Human Genetics and Biozentrum, University Würzburg, Würzburg, Germany
  • 5Institute for Clinical Genetics, Technische Universität Dresden, Dresden, Germany
  • 6Department of Congenital Heart Disease and Pediatric Cardiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
  • 7Department of Obstetrics and Gynecology, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany
  • 8Institute of Human Genetics, University of Bonn, Bonn, Germany
  • 9Department of Genomics, Life & Brain Center, University of Bonn, Bonn, Germany
  • 10Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Kiel, Germany
  • 11Institute of Epidemiology and Biobank PopGen, Christian-Albrechts-University of Kiel, Kiel, Germany
  • 12Cologne Center for Genomics, University of Cologne, Cologne, Germany
  • 13Institute of Human Genetics, University of Cologne, Cologne, Germany
  • 14Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, Cologne, Germany
  • 15Department for Gynecology and Obstetrics, LMU Munich, Munich, Germany
  • 16German Center for Neurodegenerative Diseases, Bonn, Germany
  • 17Department of Psychiatry and Psychotherapy, University of Cologne, Cologne, Germany
  • 18Department of Psychiatry and Psychotherapy, University of Bonn, Bonn, Germany
  • 19Institute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany
JAMA Oncol. 2017;3(9):1245-1248. doi:10.1001/jamaoncol.2016.5592
Key Points

Question  Do inactivating germline mutations within the FANCM gene increase breast cancer and/or ovarian cancer risk?

Findings  This case-control study included 2047 BRCA1 and BRCA2–negative familial breast cancer cases and 2187 controls and revealed an association of FANCM mutations with breast cancer. More pronounced associations were identified for early-onset (before age 51 years) breast cancer and triple-negative breast cancer. Analysis of 628 unselected ovarian cancer cases revealed no significant association.

Meaning  We suggest FANCM be included in diagnostic gene panel testing for individual breast cancer risk assessment.

Abstract

Importance  Germline mutations in established moderately or highly penetrant risk genes for breast cancer (BC) and/or ovarian cancer (OC), including BRCA1 and BRCA2, explain fewer than half of all familial BC and/or OC cases. Based on the genotyping of 2 loss-of-function (LoF) variants c.5101C>T (p.GIn1701Ter [rs147021911]) and c.5791C>T (p.Arg1931Ter [rs144567652]), the FANCM gene has been suggested as a novel BC predisposition gene, while the analysis of the entire coding region of the FANCM gene in familial index cases and geographically matched controls is pending.

Objectives  To assess the mutational spectrum within the FANCM gene, and to determine a potential association of LoF germline mutations within the FANCM gene with BC and/or OC risk.

Design, Setting, and Participants  For the purpose of identification and characterization of novel BC and/or OC predisposition genes, a total of 2047 well-characterized familial BC index cases, 628 OC cases, and 2187 geographically matched controls were screened for LoF mutations within the FANCM gene by next-generation sequencing. All patients previously tested negative for pathogenic BRCA1 and BRCA2 mutations. All data collection occurred between June 1, 2013, and April 30, 2016. Data analysis was performed from May 1, 2016, to July 1, 2016.

Main Outcomes and Measures  FANCM LoF mutation frequencies in patients with BC and/or OC were compared with the FANCM LoF mutation frequencies in geographically matched controls by univariate logistic regression. Positive associations were stratified by age at onset and cancer family history.

Results  In this case-control study, 2047 well-characterized familial female BC index cases, 628 OC cases, and 2187 geographically matched controls were screened for truncating FANCM alterations. Heterozygous LoF mutations within the FANCM gene were significantly associated with familial BC risk, with an overall odds ratio (OR) of 2.05 (95% CI, 0.94-4.54; P = .049) and a mutation frequency of 1.03% in index cases. In familial patients whose BC onset was before age 51 years, an elevated OR of 2.44 (95% CI, 1.08-5.59; P = .02) was observed. A more pronounced association was identified for patients with a triple-negative BC tumor phenotype (OR, 3.75; 95% CI, 1.00-12.85; P = .02). No significant association was detected for unselected OC cases (OR, 1.74; 95% CI, 0.57-5.08; P = .27).

Conclusions and Relevance  Based on the significant associations of heterozygous LoF mutations with early-onset or triple-negative BC, FANCM should be included in diagnostic gene panel testing for individual risk assessment. Larger studies are required to determine age-dependent disease risks for BC and to assess a potential role of FANCM mutations in OC pathogenesis.

Introduction

Monoallelic mutations within several Fanconi anemia complementation group genes, including FANCD1/BRCA2, FANCJ/BRIP1, FANCO/RAD51C, and FANCN/PALB2, confer a moderate to high risk for breast cancer (BC) and/or ovarian cancer (OC),1-3 while the role of other Fanconi anemia–associated genes in BC and/or OC pathogenesis remains elusive. Among those genes, FANCM (OMIM 609644) is a plausible candidate because the FANCM protein and its binding partner FAAP24 (OMIM 610884) are essentially required to anchor the multi-subunit Fanconi anemia core complex to chromatin after DNA damage.4 So far, 2 truncating germline variants within the FANCM gene, c.5101C>T (p.GIn1701Ter [rs147021911]) and c.5791C>T (p.Arg1931Ter [rs144567652]), have been associated with an increased BC risk.5-7

A study in the Finnish population revealed the nonsense variant c.5101C>T to be associated with familial BC.6 With a carrier frequency (CF) of 1.83% (38 in 2080) in female control individuals, this variant appears to be particularly frequent in the Finnish population. Overall, the c.5101C>T mutation was found in 3.39% (45 in 1327) of BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185)–negative familial BC cases (odds ratio [OR], 2.11; 95% CI, 1.34-3.32; P = .001) with an even higher CF of 5.88% (12 in 204) observed in a subgroup of mainly unselected cases with a triple-negative (TN) BC tumor phenotype (OR, 3.56; 95% CI, 1.81-6.98; P < .001). No significant correlation was established in 548 unselected OC cases (OR, 1.56; 95% CI, 0.75-3.26; P = .23). A multinational study investigating c.5791C>T, the second variant shown to be associated with BC, in 8635 BRCA1 and BRCA2–negative familial BC cases and 6625 control individuals confirmed an association between truncating FANCM mutations and BC risk, with a CF of 0.21% (18 in 8635) in cases and 0.06% (4 in 6625) in controls and an OR of 3.93 (95% CI, 1.28-12.11; P = .02).7 The c.5791C>T variant has shown to create an exonic splicing silencer, resulting in incomplete exon 22 skipping and protein truncation (p.Gly1906Alafs12Ter). Thus, the c.5791C>T nonsense variant is only barely detectable on transcript level.7

Because all studies described to date rely on only 2 truncating germline variants, mutational analysis of the entire coding region of the FANCM gene in patients and geographically matched controls is pending. Moreover, a higher cumulative number of patients known to carry truncating FANCM mutations is required to establish genotype-phenotype correlations, especially regarding the TNBC tumor phenotype and cancer site.

Methods

In the course of a whole exome sequencing approach, we identified the c.5101C>T germline mutation in a BRCA1 and BRCA2–negative familial BC index case from a high-risk family of German origin, which prompted us to analyze the FANCM gene in detail. The mutation was also present in the affected mother and maternal aunt, both affected by BC (eFigure in the Supplement). Based on the whole exome sequencing data provided by the Exome Aggregation Consortium, 0.65% of more than 22000 individuals of non-Finnish European origin carried heterozygous loss-of-function (LoF) mutations within the FANCM gene (excluding The Cancer Genome Atlas data; eTable 1 in the Supplement). To assess the cumulative CF of FANCM in the German population, we analyzed the whole exome sequencing data of a cohort of 2187 independent individuals not enriched for cancer phenotypes. Concordant with the data provided by the Exome Aggregation Consortium, 11 in 2187 controls carried heterozygous germline LoF mutations, resulting in a cumulative CF of 0.50% (eTable 1 in the Supplement).

Between May 1, 2016, and July 1, 2016, we analyzed the entire coding region of the FANCM gene in a well-characterized cohort of 2047 BRCA1 and BRCA2–negative familial index patients with BC of German origin by focusing on LoF alterations. Family history was considered positive when the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for genetic germline testing are fulfilled (eTable 2 in the Supplement).

Written informed consent was obtained from all patients, and ethical approval was given by the Ethics Committee of the University of Cologne.

Results

Overall, 21 in 2047 index cases carried heterozygous truncating FANCM mutations, resulting in a cumulative CF of 1.03% (OR, 2.05; 95% CI, 0.94-4.54; P = .049), as compared with the whole exome sequencing data from German controls (Table). A total of 1547 (76%) in 2047 patients were affected by BC and reported no personal or family history of OC. Among these 1547, we identified truncating mutations in 16 patients, resulting in a cumulative mutation frequency of 1.03%. Of the remaining 500 patients with BC who had either a personal history (62 [12.4%]) or family history (438 [87.6%]) of OC, 5 (1.00%) carried truncating FANCM alterations. In addition to the 62 familial index cases diagnosed with BC and OC, we subsequently analyzed 628 BRCA1 and BRCA2–negative OC cases not selected for family history. Comparable with the data reported by Kiiski et al,6 a marginally increased cumulative CF of 0.870% (6 in 690) was observed in OC cases vs controls (OR, 1.74; 95% CI, 0.57-5.08; P = .27), although the differences did not reach statistical significance. In summary, this first investigation of the entire coding sequence of the FANCM gene revealed a weak but significant overall association with familial BC.

When we stratified the 2047 familial BC index cases for age at onset (AAO), 1393 (68%) cases had an AAO before 51 years. In this subgroup (mean AAO, 41 years), 17 mutation carriers were identified, resulting in a cumulative CF of 1.22% (17 in 1393) (OR, 2.44; 95% CI, 1.08-5.59; P = .02). In patients with BC whose AAO was 51 years or older (mean AAO, 59 years), the cumulative CF was 0.613% (4 in 653), which is similar to that observed in controls. Based on genotyping of 204 mainly unselected TNBC cases, Kiiski et al6 reported a significant association between the FANCM nonsense variant c.5101C>T with the TNBC tumor phenotype in the Finnish population (OR, 3.56; 95% CI, 1.81-6.98; P < .001). In our cohort of 2047 familial BC index cases, a TNBC tumor phenotype was reported in 215 (10.50%) cases, of which 4 carried a heterozygous FANCM mutation (CF, 1.86% [4 in 215]; OR, 3.75; 95% CI, 1.00-12.85; P = .02). Thus, our results confirm the association of heterozygous LoF mutations within the FANCM gene with the TNBC tumor phenotype and establish an association with an early-onset BC disease in familial index cases. In total, we identified 21 index patients with BC who carried heterozygous LoF mutations within the FANCM gene. In this cohort of 21 mutation carriers, we observed a mean AAO of BC as 46 years (age range, 32-65 years). One mutation carrier also developed serous OC at age 62 years. Most BC cases are of no special type and classified as grade 2 (eTable 3 in the Supplement). Three additional affected mutation carriers were identified by segregation analysis in 2 families (eTable 3 and eFigure in the Supplement).

Discussion

Screening the entire coding sequence of the FANCM gene revealed its weak but overall significant association with familial BC. The study established an association between FANCM and early-onset BC disease in familial index cases. In addition, it confirmed an association of heterozygous LoF mutations within the FANCM gene with the TNBC tumor phenotype. However, this study focused on familial cases, which may lead to a significant selection bias in determining mutation frequency. For other moderately penetrant risk genes, such as CHEK2, it has been demonstrated that ORs are higher in familial than in unselected BC cases.8 Thus, it might be worthwhile to analyze unselected BC cases and then stratify for early-onset BC or TNBC. Moreover, additional studies will be required in larger sample sets to establish a potential role of FANCM mutations in OC pathogenesis. Nevertheless, based on the present analyses and previously published findings,5-7 we recommend including FANCM in diagnostic gene panel testing.

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Article Information

Accepted for Publication: September 30, 2016.

Corresponding Author: Eric Hahnen, PhD, Center for Molecular Medicine Cologne, University Hospital Cologne, Kerpener Straße 34, 50931 Cologne, Germany (eric.hahnen@uk-koeln.de).

Published Online: December 29, 2016. doi:10.1001/jamaoncol.2016.5592

Author Contributions: Dr Hahnen had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Mr Neidhardt and Dr Hauke shared first authorship.

Study concept and design: Hahnen.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Neidhardt, Meindl, Schmutzler, Hahnen, Hauke.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Engel, Ernst, Neidhardt.

Obtained funding: Schmutzler.

Study supervision: Hahnen, Schmutzler.

Conflict of Interest Disclosures: None reported.

Funding/Support: This study was supported by the German Cancer Aid grant 109076. Whole exome sequencing data were analyzed on a high-performance cluster of the Regional Computing Center of Cologne, Germany, with aid from a grant by the Deutsche Forschungsgemeinschaft.

Role of the Funder/Sponsor: The funding source had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We are very thankful to all family members who participated in this study.

References
1.
Easton  DF, Pharoah  PD, Antoniou  AC,  et al.  Gene-panel sequencing and the prediction of breast-cancer risk.  N Engl J Med. 2015;372(23):2243-2257.PubMedGoogle ScholarCrossref
2.
Meindl  A, Hellebrand  H, Wiek  C,  et al.  Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as a human cancer susceptibility gene.  Nat Genet. 2010;42(5):410-414.PubMedGoogle ScholarCrossref
3.
Antoniou  AC, Casadei  S, Heikkinen  T,  et al.  Breast-cancer risk in families with mutations in PALB2 N Engl J Med. 2014;371(6):497-506.PubMedGoogle ScholarCrossref
4.
Ciccia  A, Ling  C, Coulthard  R,  et al.  Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM.  Mol Cell. 2007;25(3):331-343.PubMedGoogle ScholarCrossref
5.
Gracia-Aznarez  FJ, Fernandez  V, Pita  G,  et al.  Whole exome sequencing suggests much of non-BRCA1/BRCA2 familial breast cancer is due to moderate and low penetrance susceptibility alleles.  PLoS One. 2013;8(2):e55681.PubMedGoogle ScholarCrossref
6.
Kiiski  JI, Pelttari  LM, Khan  S,  et al.  Exome sequencing identifies FANCM as a susceptibility gene for triple-negative breast cancer.  Proc Natl Acad Sci U S A. 2014;111(42):15172-15177.PubMedGoogle ScholarCrossref
7.
Peterlongo  P, Catucci  I, Colombo  M,  et al.  FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor.  Hum Mol Genet. 2015;24(18):5345-5355.PubMedGoogle ScholarCrossref
8.
Cybulski  C, Wokołorczyk  D, Jakubowska  A,  et al.  Risk of breast cancer in women with a CHEK2 mutation with and without a family history of breast cancer.  J Clin Oncol. 2011;29(28):3747-3752.PubMedGoogle ScholarCrossref
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