Evaluation of Fanconi Anemia Genes in Familial Breast Cancer Predisposition

[CANCER RESEARCH 63 8596–8599, December 15, 2003] Advances in Brief

Sheila Seal,1 Rita Barfoot,1 Hiran Jayatilake,1 Paula Smith,2 Anthony Renwick,1 Linda Bascombe,1 Lesley McGuffog,2 D. Gareth Evans,3 Diana Eccles,4 The Breast Cancer Susceptibility Collaboration (UK), Douglas F. Easton,2 Michael R. Stratton,1,5 and Nazneen Rahman1 1Section of Cancer Genetics, Institute of Cancer Research, Sutton, Surrey; 2Cancer Research UK Genetic Epidemiology Unit, Strangeways Research Laboratories, University of Cambridge, Cambridge; 3Department of Medical Genetics, St Mary’s Hospital, Manchester; 4Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton; and 5Cancer Genome Project, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambs, United Kingdom

Abstract underlying FA-A, FA-C, FA-E, FA-F, and FA-G were isolated by functional complementation of mitomycin-C-sensitive cells with Fanconi Anemia (FA) is an autosomal recessive syndrome character- cDNAs. FANCA was also identified by positional cloning, as was ized by congenital abnormalities, progressive bone marrow failure, and FANCD2 (reviewed in Refs. 1, 2). susceptibility to cancer. FA has eight known complementation groups and is caused by mutations in at least seven genes. Biallelic BRCA2 mutations The proteins encoded by FA genes are intimately related to each were shown recently to cause FA-D1. Monoallelic (heterozygous) BRCA2 other in molecular pathways involved in DNA repair, and FANCA, mutations confer a high risk of breast cancer and are a major cause of FANCC, FANCE, FANCF, and FANCG interact directly to form a familial breast cancer. To investigate whether heterozygous variants in multisubunit nuclear complex (3). In response to DNA damage this other FA genes are high penetrance breast cancer susceptibility alleles, we complex is translocated to DNA repair foci containing BRCA1 and screened germ-line DNA from 88 BRCA1/2-negative families, each with at BRCA2 (4). The convergence of FA gene and BRCA1/BRCA2 bio- least three cases of breast cancer, for mutations in FANCA, FANCC, logical pathways (5), the fact that cell lines homozygous for BRCA1 FANCD2, FANCE, FANCF, and FANCG. Sixty-nine sequence variants or BRCA2 mutations are hypersensitive to mitomycin-C (6, 7), and the were identified of which 25 were exonic. None of the exonic variants observation that homozygous BRCA2 mutant mice have phenotypic resulted in translational frameshifts or nonsense codons and 14 were polymorphisms documented previously. Of the remaining 11 exonic vari- features similar to FA (8), led Howlett et al. (9) to screen FA cases ants, 2 resulted in synonymous changes, and 7 were present in controls. without mutations in known FA genes for mutations in BRCA1 and Only 2 conservative missense variants, 1 in FANCA and1inFANCE, were BRCA2. One FA-B and two unassigned FA cases were each heterozy- each found in a single family and were not present in 300 controls. The gous for truncating BRCA2 mutations. A second BRCA2 sequence results indicate that FA gene mutations, other than in BRCA2, are unlikely variant of unknown significance was identified in each case (9). The to be a frequent cause of highly penetrant breast cancer predisposition. reference FA-D1 cell line was homozygous for a BRCA2 splicing mutation that results in an in-frame deletion of four amino acids, and Introduction an additional FA-D1 case carried two truncating BRCA2 mutations Fanconi Anemia (FA) is a rare autosomal recessive syndrome with (9). Overall, these data suggest strongly that the gene causing FA-D1 a prevalence of about 1–5 per million and a heterozygote frequency is BRCA2. This is additionally supported by the observation of partial estimated at 1 in 300 (for all FA disease alleles together) in Europe rescue of sensitivity to mitomycin C of a FA-D1 cell line by intro- and the United States. FA is characterized clinically by skeletal duction of wild-type BRCA2 (9). abnormalities, skin pigmentary defects, and short stature together with The majority of site-specific breast cancer families with three, four, progressive bone marrow failure, cancer susceptibility, and cellular or five cases diagnosed earlier than age 60 are not due to BRCA1 or hypersensitivity to DNA cross-linking agents, such as mitomycin C BRCA2 (10). Moreover, no more than 20% of the familial risk of and cisplatin. Somatic cell fusion analyses led to FA cases being breast cancer is accounted for by currently recognized breast cancer assigned into eight distinct complementation groups, FA-A, FA-B, susceptibility genes, BRCA1, BRCA2, TP53, PTEN, ATM, and FA-C, FA-D1, FA-D2, FA-E, FA-F, and FA-G. FA-A is the most CHEK2 (11). Therefore, the identification of BRCA2 mutations in common FA subtype in most populations, accounting for ϳ65% of FA-D1 patients has fostered speculation that heterozygotes for muta- cases; FA-C and FA-G each account for approximately 10–15% of tions in other FA genes may have a high risk of breast cancer, similar cases, with the remaining subtypes being rare (Table 1). The genes to that seen in BRCA2 heterozygotes. Homozygotes for FA gene mutations are clearly at increased risk of cancer, because FA patients Received 7/4/03; revised 9/19/03; accepted 10/24/03. often develop acute myeloid leukemia in childhood, and those that Grant support: Cancer Research UK. survive to adulthood are at increased risk of developing solid tumors, The costs of publication of this article were defrayed in part by the payment of page especially hepatic adenomas and squamous cell carcinomas of the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. esophagus, oropharynx, and vulva (12). Epidemiological studies have Notes: The Breast Cancer Susceptibility Collaboration (UK) members that supplied not detected increased cancer risks in FA heterozygotes (13, 14). families: Audrey Ardern-Jones, Rachel Belk, Nicola Bradshaw, Angela Brady, Barbara Bullman, Roseanne Cetnarsryj, Cyril Chapman, Trevor Cole, Gillian Crawford, Carol However, these studies have been limited by their small sample sizes Cummings, Rosemarie Davidson, Alan Donaldson, Diana Eccles, Rosalind Eeles, Gareth and their inability to study individual complementation groups. Evans, Sheila Goff, Jonathon Gray, Helen Gregory, Neva Haites, Shirley Hodgson, Tessa Homfray, Richard Houlston, Louise Izatt, Liane Jackson, Lisa Jeffers, Fiona Lalloo, Mark Hence, anecdotal reports of clustering of cancer cases in FA families Longmuir, Donna McBride, James Mackay, Alex Magee, Sahar Mansour, Patrick Morrison, and reported nonsignificant increases in bladder, breast cancer, and Vicky Murday, Joan Paterson, Mary Porteous, Nazneen Rahman, Mark Rogers, Andy gastric cancer remain interesting and require additional consideration Schofield, Sue Shanley, Janet Shea-Simmonds, and Lesley Snadden; Supplementary data for this article are available at Cancer Research Online (http://canceres.aacrjournals.org). (13, 15). Requests for reprints: Michael R. Stratton, The Cancer Genome Project, The To evaluate directly whether sequence variants of FA genes other Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambs, CB10 1SA, United Kingdom. Phone: 44-1223-494-951; Fax: 44-1223-494-969; E-mail: than FANCD1/BRCA2 might be rare, high-risk breast cancer suscep- [email protected]. tibility alleles, we have screened the coding sequence and intron-exon 8596

Table 1 Fanconi anemia (FA) complementation groups and genes Estimated proportion Chromosomal No. of fragments FA group Gene FA patients location No. of exons screened FA-A FANCA 66% 16q24.3 43 41 FA-B poss BRCA2 Ͻ1% FA-C FANCC 12% 9q22.3 14 14 FA-D1 BRCA2 Ͻ1% 13q12 27 FA-D2 FANCD2 Ͻ1% 3p25.3 44 43 FA-E FANCE 4% 6p21.3 10 11 FA-F FANCF 4% 11p15 1 5 FA-G FANCG/XRCC9 12% 9p13 14 14 boundaries of all of the FA genes in BRCA1/2-negative breast cancer FANCD2, FANCE, FANCF, and FANCG. Each family was charac- families from the United Kingdom. terized by at least three cases of breast cancer diagnosed under age 60. At least two (and usually three) of these early onset cases were first- Materials and Methods or second-degree relatives. The distribution of breast cancer cases per Ascertainment of Cases and Controls. Breast cancer families with at least family was as follows: 8 cases, 1; 7 cases, 3; 6 cases, 8; 5 cases,18; 4 three cases of breast cancer were ascertained as part of the Familial Breast cases, 28; and 3 cases, 30. The number of breast cancer cases diag- Cancer Study from Clinical Genetics centers in the United Kingdom, with the nosed under age 60 per family was: 7 cases, 1; 6 cases, 2; 5 cases, 7; approval of the London Multi-Research Ethics Committee. Healthy controls 4 cases, 22; and 3 cases, 56. were obtained from Human Random Control DNA panels from the European In total we identified 69 sequence variants in the 88 samples from Collection of Cell Cultures (Salisbury, United Kingdom). Controls were Cau- breast cancer families. Forty-four of the variants were intronic (not casians from the United Kingdom. involving consensus splice sites) and 7 (all in FANCA) have been Analyses of BRCA1 and BRCA2. DNA was extracted using standard reported as polymorphisms, i.e., sequence variants that are not asso- methods. At least one case from each family was screened through the 6 complete coding sequence of BRCA1 and BRCA2 by conformation sensitive ciated with FA (see Supplementary Data). On the basis of the likely gel electrophoresis (16) and was negative. The residual probabilities of carry- absence of effect on protein function and the fact that most were seen ing a BRCA1 or BRCA2 mutation were evaluated using the model of Antoniou in multiple individuals, we consider it unlikely that these intronic et al. (17). This algorithm models the familial aggregation of breast cancer in variants are high-risk breast cancer susceptibility alleles. Therefore, terms of mutations in BRCA1 and BRCA2 and an additional “polygenic” these were not additionally investigated. No large deletions of FANCA component representing the effects of a large number of genes of small effect. were detected in the quantitative multiplex PCR. The algorithm was implemented in the program Mendel, and the residual Twenty-six exonic variants were identified, but none caused trans- probability that the index case in each family carried a mutation was computed lational frameshift or nonsense codons, and 8 were silent variants. conditional on their family history and on their being tested for mutations in Eighteen exonic variants encoded missense changes. Nine of these BRCA1 and BRCA2. Using this algorithm the expected number of mutation were known polymorphisms, 2 have been reported as mutations, and carriers remaining in the set was 4.4 for BRCA1 and 5.9 for BRCA2. Mutation Analysis of FA Genes. FANCA, FANCC, FANCD2, FANCE, 7 were novel (Table 2). To investigate the 9 missense alterations that FANCF, and FANCG were screened for small intragenic mutations by con- were either reported as FA-causing mutations (FANCA S1088F and formation sensitive gel electrophoresis. Amplifying primers flanking exons FANCA H1417D) or were not reported previously (FANCA L1143V; and intron-exon boundaries were designed using the genomic sequence for FANCC V60I, FANCC E417L; FANCD2 T896M; FANCE R365K, each gene, and Primer3 software (primers and conditions available on request). FANCE A502T; and FANCF P320L) we screened the relevant exons The number of exons and fragments screened for each gene is shown in Table in 300 United Kingdom controls and detected 7 of the variants (Table 1. Three fragments (FANCD2 exons 14, 20, and 24) produced data that was 2). Only 2 conservative missense variants, FANCA L1143V and difficult to interpret even after redesign of the primers, and we consider these FANCE R365K, were not detected in controls and have not been exons to be unscreened. The poor quality of these fragments may be due to reported previously as polymorphisms. To additionally assess these 2 partial genomic copies of this gene. This problem might conceivably compro- variants we examined their segregation with breast cancer in the mise screening other parts of FANCD2, although we were able to detect polymorphisms reported previously and novel variants without apparent dif- families in which they were detected. FANCA L1143V was identified ficulty. Complete data from the remaining 125 fragments was obtained from in family B637, which consists of twin sisters and two additional Ͼ90% of samples. Genomic DNA from cases showing mobility shifts was sisters all affected with breast cancer before age 55 years. The twins sequenced bidirectionally using the BigDyeTerminator Cycle Sequencing kit (who are thought to be monozygotic) and one sister carried FANCA and a 3100 automated sequencer (ABI Perkin-Elmer). L1143V as did an unaffected brother. The status of the fourth sister is Approximately 40% of FANCA mutations are heterozygous large intragenic unknown. FANCE R365K was identified in family B529, which deletions. We used a quantitative fluorescent multiplex PCR assay published consists of a mother and two daughters all affected with breast cancer ϳ previously to screen for 75% of known FANCA deletions (18). In the before 60 years of age, all of whom carried the variant. multiplex, 6-fam-labeled primers for FANCA exons 5, 17, 35, and 43, and Our analyses are the first to report mutation screens of FANCD2, FANCC exons 5 and 6 (which were used as controls) were amplified simul- FANCE, and FANCF since the original discovery of these genes. We taneously by PCR. The resulting products were electrophoresed on an ABI 3100 sequencer (ABI Perkin-Elmer) and analyzed with GENOTYPER soft- have detected a number of novel sequence variants and have also ware. All of the experiments were repeated six times, and positive controls for demonstrated that two variants reported previously as causing FA are FANCA exon 5 and exon 17 deletions were included in all of the experiments. unlikely to be classical FA disease-causing mutations. FANCA A consistent, reproducible reduction by half in peak height of a FANCA exon S1088F was reported as the causative mutation in a consanguineous compared with the FANCC exons was taken as evidence of a deletion. The Middle Eastern FA-A family (19). However, our data show it is a quantitative PCR assays, particularly for exon 5, were variably successful, but relatively common variant found in healthy individuals and, therefore, evaluable data from 79 samples were obtained. perhaps more likely to be a polymorphism. FANCA H1714D has also Results and Discussion been reported as a mutation in a Middle Eastern consanguineous

DNAs from 88 breast cancer families without BRCA1 or BRCA2 6 The Fanconi Anemia Mutation Database, internet address: http://www.rockerfeller. mutations were screened for sequence variants in FANCA, FANCC, edu/fanconi/mutate. 8597

Table 2 Exonic Fanconi anemia (FA) gene sequence variants identified in this study No. of heterozygote No. of heterozygote Gene/Exon Nucleotide change Protein change cases (n ϭ 88) controls (n ϭ 300) FA database entry FANCA 13 1143TϾG T381T 8 Not tested Polymorphism 14 1235CϾT A412V 1 Not tested Polymorphism 16 1501GϾA G501S Ͼ10 Not tested Polymorphism 22 1927CϾG P643A Ͼ10 Not tested Polymorphism 23 2151GϾT M717I 6 Not tested Polymorphism 26 2462GϾA G809D Ͼ10 Not tested Polymorphism 30 2901CϾT S967S Ͼ10 Not tested Polymorphism 33 3263CϾT S1088F Ͼ10 Ͼ10 Mutation 35 3427CϾG L1143V 1 0 Not entered 35 3430CϾTa R1144W 0 1 Not entered 42 4249CϾG H1417D 2 1 Mutation 42 4186AϾGa I1396V 0 1 Not entered FANCC 2 332CϾT S26F 4 Not tested Polymorphism 3 433GϾA V60I 1 1 Not entered 7 839GϾA D195V 1 Not tested Polymorphism 8 1071CϾT I272I 1 Not tested Not entered 13 1504GϾA E417L 1 1 Not entered 14 1597GϾA V449M 1 Not tested Polymorphism FANCD2 17 1440TϾC H480H Ͼ10 Not tested Polymorphism 17 1509TϾC N503N Ͼ10 Not tested Polymorphism 23 2141CϾT L714P Ͼ10 Not tested Polymorphism 28 2687CϾT T896M Ͼ10 Ͼ10 Not entered 42 4098TϾG L1366L Ͼ10 Not tested Polymorphism FANCE 2 387CϾA P129P Ͼ10 Not tested Not entered 5 1094GϾA R365K 1 0 Not entered 5 1071CϾT L357L 3 Ͼ10 Not entered 5 1018GϾCa G349R 0 2 Not entered 5 1066AϾGa S356G 0 1 Not entered 9 1504GϾA A502T 2 Ͼ10 Not entered FANCF 1 959CϾT P320L 1 Ͼ10 Not entered a These variants were detected only in controls and were not present in the breast cancer samples.

family (20). However, we identified this variant in two cases and one quency of FANC gene sequence variants in large series of breast control, again suggesting that it is a polymorphism. These results cancer cases and healthy controls. emphasize the need for caution in interpretation of missense variants, particularly in consanguineous cases where all of the sequence alter- Acknowledgments ations in and around the relevant gene are likely to be homozygous in affected individuals. We thank all individuals and families who have participated in these studies. We thank Anita Hall and Karen Redman for family ascertainment, and Chris We have not identified any clearly pathogenic FA gene mutations Mathew for positive controls for FANCA deletions. in 88 breast cancer pedigrees. Only 2 missense coding variants, FANCA L1143V and FANCE R365K, were seen in familial breast References cancer cases and were absent in controls. Although these variants segregated with breast cancer in the families, the pedigrees are small, 1. Joenje, H., and Patel, K. J. The emerging genetic and molecular basis of Fanconi Anemia. Nat. Rev. Genet., 2: 447–457, 2001. and segregation could have occurred by chance. Moreover, both 2. Tischkowitz, M., and Hodgson, S. V. Fanconi Anemia. J. Med. Genet., 40: 1–10, amino acid changes are conservative and, hence, overall are unlikely 2003. 3. Medhurst, A. L., Huber, P. A., Waisfisz, Q., de Winter, J. P., and Mathew, C. G. to be high-penetrance breast cancer susceptibility alleles. Direct interactions of the five known Fanconi anemia proteins suggest a common In this study, we have investigated whether individual variants of functional pathway. Hum. Mol. Genet., 10: 423–429, 2001. individual FA genes may be high-risk breast cancer susceptibility 4. Garcia-Higuera, I., Taniguchi, T., Ganescan, S., Meyn, M. S., Timmers, C., Hejna, J., Grompe, M., and D’Andrea, A. D. Interaction of the Fanconi anemia proteins and alleles. Overall, our data suggest that heterozygous FA gene muta- BRCA1 in a common pathway. Mol. Cell, 7: 249–262, 2001. tions, except for those in BRCA2, do not confer a high risk of breast 5. D’Andrea, A. D., and Grompe, M. The Fanconi Anaemia/BRCA pathway. Nat. Rev. cancer and are not making a major contribution to familial breast Cancer, 3: 23–34, 2003. 6. Moynahan, M. E., Cui, T. Y., and Jain, M. Homology-directed DNA repair, mito- cancer. Our data are consistent with epidemiological studies, which mycin-c resistance, and chromosome stability is restored with correction of a Brca1 suggest that heterozygous FA mutation carriers are not at increased mutation. Cancer Res., 61: 4842–4850, 2001. 7. Patel, K. J., Yu, V. P., Lee, H., Corcoran, A., Thistletwaite, F. C., Evans, M. J., risk of breast cancer. Because we have screened only 88 BRCA1/2- Colledge, W. H., Friedman, L. S., Ponder, B. A., and Venkitaraman, A. R. Involve- negative breast cancer families it remains possible that some FA gene ment of Brca2 in DNA repair. Mol. Cell, 1: 347–357, 1998. mutations do confer a high risk of breast cancer, although if this were 8. Connor, F., Bertwistle, D., Mee, P. J., Ross, G. M., Swift, S., Grigorieva, E., Tybulewicz, V. L., and Ashworth, A. Tumorigenesis and a DNA repair defect in mice the case they must be rare in comparison with BRCA1 or BRCA2 with a truncating Brca2 mutation. Nat. Genet., 17: 423–430, 1997. mutations. (By way of comparison, a family set of this type would be 9. Howlett, N. G., Taniguchi, T., Olson, S., Cox, B., Waisfisz, Q., De Die-Smulders, C., expected to contain ϳ20 deleterious BRCA1 or BRCA2 mutations). Persky, N., Grompe, M., Joenje, H., Pals, G., Ikeda, H., Fox, E. A., and D’Andrea, A. D. Biallelic inactivation of BRCA2 in Fanconi Anemia. Science (Wash. DC), 297: However, our current analyses do not exclude the possibility that FA 606–609, 2002. gene sequence variants are associated with low penetrance breast 10. Ford, D., Easton, D. F., Stratton, M., Narod, S., Goldgar, D., Devilee, P., Bishop, D. T., Weber, B., Lenoir, G., Chang-Claude, C., Sobol, H., Teare, M. D., Struewing, cancer susceptibility similar to CHEK2 1100delC (21). Evaluation of J., Arason, A., Scherneck, S., Peto, J., Rebbeck, T. R., Tonin, P., Neuhausen, S., this hypothesis will require additional studies comparing the fre- Barkardottir, R., Eyfjord, J., Lynch, H., Ponder, B. A. J., Gayther, S. A., Birch, J. M., 8598

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8599

Sheila Seal, Rita Barfoot, Hiran Jayatilake, et al.

Cancer Res 2003;63:8596-8599.

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