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2 mutation-negative high-risk Russian breast cancer patients
ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■
Contents lists available at ScienceDirect
Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients Anna P. Sokolenko a,b, Elena V. Preobrazhenskaya a,b, Svetlana N. Aleksakhina a, Aglaya G. Iyevleva a,b, Natalia V. Mitiushkina a, Olga A. Zaitseva a, Olga S. Yatsuk a, Vladislav I. Tiurin a,b, Tatiana N. Strelkova a, Alexandr V. Togo a,b, Evgeny N. Imyanitov a,b,c,* a
N.N. Petrov Institute of Oncology, St.-Petersburg 197758, Russia St.-Petersburg Pediatric Medical University, St.-Petersburg 194100, Russia c I.I. Mechnikov North-Western Medical University, St-Petersburg 191015, Russia b
A R T I C L E
I N F O
Article history: Received 9 December 2014 Received in revised form 18 January 2015 Accepted 19 January 2015 Keywords: Breast cancer Hereditary predisposition WRN Candidate genes
A B S T R A C T
Twenty one DNA repair genes were analyzed in a group of 95 BC patients, who displayed clinical features of hereditary disease predisposition but turned out to be negative for mutations in BRCA1 and BRCA2 entire coding region as well as for founder disease-predisposing alleles in CHEK2, NBN/NBS1 and ATM genes. Full-length sequencing of CHEK2 and NBN/NBS1 failed to identify non-founder mutations. The analysis of TP53 revealed a woman carrying the R282W allele; further testing of additional 108 BC patients characterized by a very young age at onset (35 years or earlier) detected one more carrier of the TP53 germ-line defect. In addition, this study confirmed non-random occurrence of PALB2 truncating mutations in Russian hereditary BC patients. None of the studied cases carried germ-line defects in recently discovered hereditary BC genes, BRIP1, FANCC, MRE11A and RAD51C. The analysis of genes with yet unproven BC-predisposing significance (BARD1, BRD7, CHEK1, DDB2, ERCC1, EXO1, FANCG, PARP1, PARP2, RAD51, RNF8, WRN) identified single women carrying a protein-truncating allele, WRN R1406X. DNA sequencing of another set of 95 hereditary BC cases failed to reveal additional WRN heterozygous genotypes. Since WRN is functionally similar to the known BC-predisposing gene, BLM, it deserves to be analyzed in future hereditary BC studies. Furthermore, this investigation revealed a number of rare missense germ-line variants, which are classified as probably protein-damaging by online in silico tools and therefore may require further consideration. © 2015 Published by Elsevier Ireland Ltd.
Introduction At least 5–20% of breast cancer (BC) incidence is attributed to inheritance of rare germ-line defects. BRCA1 and BRCA2 genes are among the best known genes for familial BC being detected in almost all ethnic groups. Several other DNA repair genes have been shown to be associated with hereditary BC risk, including CHEK2, NBN/ NBS1, PALB2, BRIP1, BLM etc., however their contribution to BC incidence appears to be less pronounced as compared to BRCA1 and BRCA2. For the time being, genetic analysis for known BC genes fails to identify the causative mutation in approximately two thirds of high-risk BC cases, therefore many novel hereditary BC genes remain to be identified [1–4]. Early genetic studies of familial BC were based on linkage analysis of extensive pedigrees with multiple affected women. Given the rarity of large well-described families, many researchers later opted
* Corresponding author. Tel.: +7 812 4399528; fax: +7 812 5968947. E-mail address: [email protected] (E.N. Imyanitov).
for the study of so-called candidate genes in patients with clinical features of familial BC, focusing mainly on sequencing of participants of DNA repair [1–4]. These approaches are now being gradually replaced by whole exome and whole genome sequencing [5–8]. Nevertheless, use of the next generation sequencing technologies still remains compromised by high costs, uncertainty regarding false negative rates and limitations in bioinformatic analysis, so the testing of candidate genes may not be yet considered as an entirely outdated approach. Most contemporary genetics research has been carried out in the countries of the Western world. The analysis of less comprehensively studied populations is likely to reveal new disease-causing genes, given that every ethnicity has its own ancestors and therefore unique pool of germ-line mutations. Slavic countries are of particular interest in this respect: somewhat surprisingly, they happened to preserve remarkable genetic homogeneity, so that the Eastern Slavs apparently represent the largest known founder community in the world consisting of over 200 millions of people [9]. We have previously reported the identification of a novel breast cancer predisposing gene, BLM, using a candidate gene
http://dx.doi.org/10.1016/j.canlet.2015.01.022 0304-3835/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Anna P. Sokolenko, et al., Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.01.022
ARTICLE IN PRESS A.P. Sokolenko et al./Cancer Letters ■■ (2015) ■■–■■
2
sequencing of genetically enriched Russian BC cases [10]. Here we describe further results of analysis of selected DNA repair genes in DNA samples obtained from patients with young-onset and/or bilateral and/or familial BC. Materials and methods We considered BC patients, who were selected on the basis of clinical signs of hereditary disease (Supplementary Table S1 and Fig. S1). To exclude cases with known cause of disease predisposition, we performed analysis of the entire coding region of BRCA1 or BRCA2 genes as well as genotyping for Slavic founder mutations (CHEK2: 1100delC [c.1100delC], del5395 [del ex9-10], ivs2+1G>A [c.444 + 1G>A]; NBN/ NBS1: 657del5 [c.657_661del]; ATM: c.5932G>T) [9]. The selection of gene candidates was based on prior findings or potential role in DNA repair (Supplementary Table S2). First round of testing involved high-resolution melting and sequencing analysis of 95 BC cases; primers for PCR amplification of gene fragments are described in Supplementary Table S3. Those genes, which showed promising results, were subjected to the 2nd round of analysis in another group of genetically enriched BC patients (Supplementary Table S4). The primary goal of the study was to identify germ-line mutations with clear protein-inactivating consequences, such as frame-shifts, premature stop-codons, and splice site alterations. In addition, we considered missense mutations depending on their potential functional impact. Other relevant details are described in our earlier report [10].
Results Recurrent mutations in CHEK2 and NBN/NBS1 genes play an essential role in Russian BC incidence, being detected in approximately 3% and 1% of BC, respectively [9]. We expected that full-length sequencing of these genes would identify non-founder mutations in the mentioned loci. However, no truncating mutations were identified upon the analysis of genetically enriched 95 cases. We further considered genes with already known BC-predisposing roles, which have not been yet comprehensively studied in a Russian population. For example, we have previously reported two PALB2 mutations (Q921X and R414X) in a sample consisting of 45 cases of bilateral BC [11]. Here we analyzed 120 additional genetically enriched BC, and revealed another 2 mutation carriers (c.1592delT and c.1317delG). Germ-line mutations in TP53 have initially been discovered in patients with very severe hereditary cancer disorder, i.e. Li– Fraumeni syndrome. Subsequent studies revealed that, for yet unknown reasons, the manifestation of inborn TP53 defects may be limited in selected women by breast cancer development. The analysis of an initial sample set of 95 genetically enriched BC patients revealed 1 patient with TP53 mutation (R282W). This woman was 44 years old, suffered from the ER+/PgR+/HER2+ disease, and reported BC in the mother, pancreatic cancer in the sister, and gastric cancer in the maternal grandfather. We considered evidence for
association of TP53 germ-line mutations with particularly young disease onset, and analyzed all available BC patients aged ≤35 years (n = 108). This analysis led to the identification of an additional patient with TP53 inherited defect, who suffered from bilateral cancer and had ER+/PgR+/HER2− receptor phenotype in both tumors (Supplementary Table S5). Recent studies provided evidence on BC-predisposing role of BRIP1, RAD51C, FANCC and MRE11A germ-line mutations (Supplementary Table S2). Based on established significance of these genes for hereditary BC, we included them into the testing panel. However, no truncating mutations have been identified. We further focused on genes with an unproven role in BC predisposition, which participate in key components of DNA repair (Supplementary Table S2). This effort led to the identification of a recurrent BRCA1-associated mutation in the BLM gene, which has been described in detail in our earlier report [10]. The analysis of a similar gene, WRN, also revealed an instance of heterozygous truncating mutation (R1406X); however, in contrast to BLM, the fulllength sequence examination of the extended series of genetically enriched BC failed to detect additional WRN-heterozygous patients. Other candidate genes, including BARD1, BRD7, CHEK1, DDB2, ERCC1, EXO1, FANCG, PARP1, PARP2, RAD51, RNF8, did not contain protein-truncating mutations in the analyzed BC set. In addition, a number of probably damaging missense mutations have been identified; however none of them turned out to be recurrent (Table 1). Discussion The results of this study may have some implications for breast cancer research and management. In particular, this report confirmed a noticeable occurrence of PALB2 mutations in Slavic BC patients. Given convincing evidence for potential clinical utility of PALB2 testing [19], the status of this gene has to be considered upon routine hereditary BC diagnostic activities. Also, we confirmed nonrandom occurrence of TP53 germ-line mutations in BC patients with clinical features of familial disease predisposition. It remains to be investigated why some TP53 mutation carriers experience multiple primary neoplasms starting from adolescence, while others remain cancer-free at least until midlife and eventually develop single-site breast cancer disease. Interestingly, our study failed to identify novel lesions in well-known BC-predisposing genes, CHEK2 and NBN/NBS1, although recent multigene sequencing efforts revealed that the spectrum of mutations in these genes is not limited to founder alleles [20]. Apart from previously published data on BCpredisposing significance of the BLM Q548X allele [10], we were able
Table 1 Germ-line mutations in DNA repair genes with proven or potential breast cancer predisposing role. Gene
Number of samples analyzed
Truncating or splice site mutations
TP53 TP53 PALB2 PALB2 PALB2 PALB2 BRIP1 BLM BLM EXO1 FANCC NBN/NBS1 NBN/NBS1 PARP1 PARP1 WRN WRN
203 203 165 165 165 165 95 190 95 95 95 95 95 95 95 190 190
c.560-2A>G (n = 1)
Missense variants with presumably pathogenic role
Polyphen-2 prediction
R282W (n = 1)
Probably damaging
p.R173C (n = 1)
Probably damaging
p.R1139P (n = 1) p.N279S (n = 1) p.S26F (n = 1) p.I171V (n = 1) p.R215W (n = 1) p.Q150H (n = 1) p.R857Q (n = 1)
Possibly damaging Probably damaging Probably damaging Probably damaging Probably damaging Probably damaging Probably damaging
p.D143H (n = 1)
Probably damaging
R414X (n = 1) Q921X (n = 1) c.1592delT (n = 1) c.1317delG (n = 1) Q548X (n = 2)
R1406X (n = 1)
References http://p53.iarc.fr/TP53GeneVariations.aspx rs28934574; http://p53.iarc.fr/TP53GeneVariations.aspx [11,13] [11] [14] [12] rs4988345 [10,15] This study rs4149909 [16] [17,18] [18] rs142376976 rs190105316 rs11574410 This study
Please cite this article in press as: Anna P. Sokolenko, et al., Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.01.022
ARTICLE IN PRESS A.P. Sokolenko et al./Cancer Letters ■■ (2015) ■■–■■
to identify only 1 sample with truncating mutation, WRN R1406X. Given strong functional similarity between BLM and WRN, the latter gene has to be included in the hereditary BC research agenda, although we could not demonstrate additional instances of the WRN involvement. Interestingly, a number of case–control studies provided evidence for an association between WRN gene polymorphisms and breast cancer risk [21–24]. While the mutations leading to frame-shifts, premature stopcodons or alterations in the splice sites are always considered in candidate hereditary cancer gene studies, the evaluation of significance of missense variants remains significantly more complicated. It is self-explanatory that while truncating mutations of the same protein can usually be pooled together and analyzed as a whole, each amino acid substitution needs to be considered on an individual basis. The existing tools for mutation analysis, such as in silico prediction of potential influence on protein structure, tracking of segregation with the disease in extended pedigrees, comparison of frequencies in cases vs. controls, or direct functional studies with allelic protein variants, are not sufficiently robust and fail to provide conclusive information in most of instances. Rapid accumulation of data obtained by massive parallel sequencing will lead to a dramatic increase in the number of missense germ-line variants with unknown clinical significance. The development of approaches allowing reliable discrimination between disease-causing and relatively neutral amino acid substitutions appears to be a major unmet need of contemporary medical genetic research. Acknowledgements This work was supported by the Russian Scientific Fund (grant number 14-25-00111). We cordially thank Prof. William R. Miller for stimulating discussions. Conflict of interest There are no conflicts of interest in the studies reported in the paper. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.01.022. References [1] U.O. Njiaju, O.I. Olopade, Genetic determinants of breast cancer risk: a review of current literature and issues pertaining to clinical application, Breast J. 18 (2012) 436–442. [2] P. Apostolou, F. Fostira, Hereditary breast cancer: the era of new susceptibility genes, Biomed Res Int. (2013) 747318. [3] N. Bogdanova, S. Helbig, T. Dörk, Hereditary breast cancer: ever more pieces to the polygenic puzzle, Hered Cancer Clin Pract. 11 (2013) 12. [4] F.J. Couch, K.L. Nathanson, K. Offit, Two decades after BRCA: setting paradigms in personalized cancer care and prevention, Science 343 (2014) 1466–1470.
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[5] D.J. Park, F. Lesueur, T. Nguyen-Dumont, M. Pertesi, F. Odefrey, F. Hammet, et al., Rare mutations in XRCC2 increase the risk of breast cancer, Am. J. Hum. Genet. 90 (2012) 734–739. [6] E.R. Thompson, M.A. Doyle, G.L. Ryland, S.M. Rowley, D.Y. Choong, R.W. Tothill, et al., Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles, PLoS Genet. 8 (2012) e1002894. [7] F.S. Hilbers, C.M. Meijers, J.F. Laros, M. van Galen, N. Hoogerbrugge, H.F. Vasen, et al., Exome sequencing of germline DNA from non-BRCA1/2 familial breast cancer cases selected on the basis of aCGH tumor profiling, PLoS ONE 8 (2013) e55734. [8] F.J. Gracia-Aznarez, V. Fernandez, G. Pita, P. Peterlongo, O. Dominguez, M. de la Hoya, 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 8 (2013) e55681. [9] A.P. Sokolenko, N. Bogdanova, W. Kluzniak, E.V. Preobrazhenskaya, E.S. Kuligina, A.G. Iyevleva, et al., Double heterozygotes among breast cancer patients analyzed for BRCA1, CHEK2, ATM, NBN/NBS1, and BLM germ-line mutations, Breast Cancer Res. Treat. 145 (2014) 553–562. [10] A.P. Sokolenko, A.G. Iyevleva, E.V. Preobrazhenskaya, N.V. Mitiushkina, S.N. Abysheva, E.N. Suspitsin, et al., High prevalence and breast cancer predisposing role of the BLM c.1642 C > T (Q548X) mutation in Russia, Int. J. Cancer 130 (2012) 2867–2873. [11] N. Bogdanova, A.P. Sokolenko, A.G. Iyevleva, S.N. Abysheva, M. Blaut, M. Bremer, et al., PALB2 mutations in German and Russian patients with bilateral breast cancer, Breast Cancer Res. Treat. 126 (2011) 545–550. [12] C. Balia, E. Sensi, G. Lombardi, M. Roncella, G. Bevilacqua, M.A. Caligo, PALB2: a novel inactivating mutation in a Italian breast cancer family, Fam. Cancer 9 (2010) 531–536. [13] E.P. Slater, P. Langer, E. Niemczyk, K. Strauch, J. Butler, N. Habbe, et al., PALB2 mutations in European familial pancreatic cancer families, Clin. Genet. 78 (2010) 490–494. [14] H. Erkko, B. Xia, J. Nikkilä, J. Schleutker, K. Syrjäkoski, A. Mannermaa, et al., A recurrent mutation in PALB2 in Finnish cancer families, Nature 446 (2007) 316–319. [15] J. German, M.M. Sanz, S. Ciocci, T.Z. Ye, N.A. Ellis, Syndrome-causing mutations of the BLM gene in persons in the Bloom’s Syndrome Registry, Hum. Mutat. 28 (2007) 743–753. [16] L.M. Barber, H.E. McGrath, S. Meyer, A.M. Will, J.M. Birch, O.B. Eden, et al., Constitutional sequence variation in the Fanconi anaemia group C (FANCC) gene in childhood acute myeloid leukaemia, Br. J. Haematol. 121 (2003) 57–62. [17] N. Bogdanova, P. Schürmann, R. Waltes, S. Feshchenko, I.V. Zalutsky, M. Bremer, et al., NBS1 variant I171V and breast cancer risk, Breast Cancer Res. Treat. 112 (2008) 75–79. [18] K. Roznowski, D. Januszkiewicz-Lewandowska, M. Mosor, M. Pernak, M. Litwiniuk, J. Nowak, I171V germline mutation in the NBS1 gene significantly increases risk of breast cancer, Breast Cancer Res. Treat. 110 (2008) 343– 348. [19] A.C. Antoniou, S. Casadei, T. Heikkinen, D. Barrowdale, K. Pylkäs, J. Roberts, et al., Breast-cancer risk in families with mutations in PALB2, N. Engl. J. Med. 371 (2014) 497–506. [20] L. Castéra, S. Krieger, A. Rousselin, A. Legros, J.J. Baumann, O. Bruet, et al., Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes, Eur. J. Hum. Genet. 22 (2014) 1305–1313. [21] M. Wirtenberger, B. Frank, K. Hemminki, R. Klaes, R.K. Schmutzler, B. Wappenschmidt, et al., Interaction of Werner and Bloom syndrome genes with p53 in familial breast cancer, Carcinogenesis 27 (2006) 1655–1660. [22] S.L. Ding, J.C. Yu, S.T. Chen, G.C. Hsu, C.Y. Shen, Genetic variation in the premature aging gene WRN: a case-control study on breast cancer susceptibility, Cancer Epidemiol. Biomarkers Prev. 16 (2007) 263–269. [23] P. Bhatti, J.P. Struewing, B.H. Alexander, M. Hauptmann, L. Bowen, L.H. Mateus-Pereira, et al., Polymorphisms in DNA repair genes, ionizing radiation exposure and risk of breast cancer in U.S. Radiologic technologists, Int. J. Cancer 122 (2008) 177–182. [24] Z. Wang, Y. Xu, J. Tang, H. Ma, J. Qin, C. Lu, et al., A polymorphism in Werner syndrome gene is associated with breast cancer susceptibility in Chinese women, Breast Cancer Res. Treat. 118 (2009) 169–175.
Please cite this article in press as: Anna P. Sokolenko, et al., Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.01.022
Contents lists available at ScienceDirect
Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients Anna P. Sokolenko a,b, Elena V. Preobrazhenskaya a,b, Svetlana N. Aleksakhina a, Aglaya G. Iyevleva a,b, Natalia V. Mitiushkina a, Olga A. Zaitseva a, Olga S. Yatsuk a, Vladislav I. Tiurin a,b, Tatiana N. Strelkova a, Alexandr V. Togo a,b, Evgeny N. Imyanitov a,b,c,* a
N.N. Petrov Institute of Oncology, St.-Petersburg 197758, Russia St.-Petersburg Pediatric Medical University, St.-Petersburg 194100, Russia c I.I. Mechnikov North-Western Medical University, St-Petersburg 191015, Russia b
A R T I C L E
I N F O
Article history: Received 9 December 2014 Received in revised form 18 January 2015 Accepted 19 January 2015 Keywords: Breast cancer Hereditary predisposition WRN Candidate genes
A B S T R A C T
Twenty one DNA repair genes were analyzed in a group of 95 BC patients, who displayed clinical features of hereditary disease predisposition but turned out to be negative for mutations in BRCA1 and BRCA2 entire coding region as well as for founder disease-predisposing alleles in CHEK2, NBN/NBS1 and ATM genes. Full-length sequencing of CHEK2 and NBN/NBS1 failed to identify non-founder mutations. The analysis of TP53 revealed a woman carrying the R282W allele; further testing of additional 108 BC patients characterized by a very young age at onset (35 years or earlier) detected one more carrier of the TP53 germ-line defect. In addition, this study confirmed non-random occurrence of PALB2 truncating mutations in Russian hereditary BC patients. None of the studied cases carried germ-line defects in recently discovered hereditary BC genes, BRIP1, FANCC, MRE11A and RAD51C. The analysis of genes with yet unproven BC-predisposing significance (BARD1, BRD7, CHEK1, DDB2, ERCC1, EXO1, FANCG, PARP1, PARP2, RAD51, RNF8, WRN) identified single women carrying a protein-truncating allele, WRN R1406X. DNA sequencing of another set of 95 hereditary BC cases failed to reveal additional WRN heterozygous genotypes. Since WRN is functionally similar to the known BC-predisposing gene, BLM, it deserves to be analyzed in future hereditary BC studies. Furthermore, this investigation revealed a number of rare missense germ-line variants, which are classified as probably protein-damaging by online in silico tools and therefore may require further consideration. © 2015 Published by Elsevier Ireland Ltd.
Introduction At least 5–20% of breast cancer (BC) incidence is attributed to inheritance of rare germ-line defects. BRCA1 and BRCA2 genes are among the best known genes for familial BC being detected in almost all ethnic groups. Several other DNA repair genes have been shown to be associated with hereditary BC risk, including CHEK2, NBN/ NBS1, PALB2, BRIP1, BLM etc., however their contribution to BC incidence appears to be less pronounced as compared to BRCA1 and BRCA2. For the time being, genetic analysis for known BC genes fails to identify the causative mutation in approximately two thirds of high-risk BC cases, therefore many novel hereditary BC genes remain to be identified [1–4]. Early genetic studies of familial BC were based on linkage analysis of extensive pedigrees with multiple affected women. Given the rarity of large well-described families, many researchers later opted
* Corresponding author. Tel.: +7 812 4399528; fax: +7 812 5968947. E-mail address: [email protected] (E.N. Imyanitov).
for the study of so-called candidate genes in patients with clinical features of familial BC, focusing mainly on sequencing of participants of DNA repair [1–4]. These approaches are now being gradually replaced by whole exome and whole genome sequencing [5–8]. Nevertheless, use of the next generation sequencing technologies still remains compromised by high costs, uncertainty regarding false negative rates and limitations in bioinformatic analysis, so the testing of candidate genes may not be yet considered as an entirely outdated approach. Most contemporary genetics research has been carried out in the countries of the Western world. The analysis of less comprehensively studied populations is likely to reveal new disease-causing genes, given that every ethnicity has its own ancestors and therefore unique pool of germ-line mutations. Slavic countries are of particular interest in this respect: somewhat surprisingly, they happened to preserve remarkable genetic homogeneity, so that the Eastern Slavs apparently represent the largest known founder community in the world consisting of over 200 millions of people [9]. We have previously reported the identification of a novel breast cancer predisposing gene, BLM, using a candidate gene
http://dx.doi.org/10.1016/j.canlet.2015.01.022 0304-3835/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Anna P. Sokolenko, et al., Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.01.022
ARTICLE IN PRESS A.P. Sokolenko et al./Cancer Letters ■■ (2015) ■■–■■
2
sequencing of genetically enriched Russian BC cases [10]. Here we describe further results of analysis of selected DNA repair genes in DNA samples obtained from patients with young-onset and/or bilateral and/or familial BC. Materials and methods We considered BC patients, who were selected on the basis of clinical signs of hereditary disease (Supplementary Table S1 and Fig. S1). To exclude cases with known cause of disease predisposition, we performed analysis of the entire coding region of BRCA1 or BRCA2 genes as well as genotyping for Slavic founder mutations (CHEK2: 1100delC [c.1100delC], del5395 [del ex9-10], ivs2+1G>A [c.444 + 1G>A]; NBN/ NBS1: 657del5 [c.657_661del]; ATM: c.5932G>T) [9]. The selection of gene candidates was based on prior findings or potential role in DNA repair (Supplementary Table S2). First round of testing involved high-resolution melting and sequencing analysis of 95 BC cases; primers for PCR amplification of gene fragments are described in Supplementary Table S3. Those genes, which showed promising results, were subjected to the 2nd round of analysis in another group of genetically enriched BC patients (Supplementary Table S4). The primary goal of the study was to identify germ-line mutations with clear protein-inactivating consequences, such as frame-shifts, premature stop-codons, and splice site alterations. In addition, we considered missense mutations depending on their potential functional impact. Other relevant details are described in our earlier report [10].
Results Recurrent mutations in CHEK2 and NBN/NBS1 genes play an essential role in Russian BC incidence, being detected in approximately 3% and 1% of BC, respectively [9]. We expected that full-length sequencing of these genes would identify non-founder mutations in the mentioned loci. However, no truncating mutations were identified upon the analysis of genetically enriched 95 cases. We further considered genes with already known BC-predisposing roles, which have not been yet comprehensively studied in a Russian population. For example, we have previously reported two PALB2 mutations (Q921X and R414X) in a sample consisting of 45 cases of bilateral BC [11]. Here we analyzed 120 additional genetically enriched BC, and revealed another 2 mutation carriers (c.1592delT and c.1317delG). Germ-line mutations in TP53 have initially been discovered in patients with very severe hereditary cancer disorder, i.e. Li– Fraumeni syndrome. Subsequent studies revealed that, for yet unknown reasons, the manifestation of inborn TP53 defects may be limited in selected women by breast cancer development. The analysis of an initial sample set of 95 genetically enriched BC patients revealed 1 patient with TP53 mutation (R282W). This woman was 44 years old, suffered from the ER+/PgR+/HER2+ disease, and reported BC in the mother, pancreatic cancer in the sister, and gastric cancer in the maternal grandfather. We considered evidence for
association of TP53 germ-line mutations with particularly young disease onset, and analyzed all available BC patients aged ≤35 years (n = 108). This analysis led to the identification of an additional patient with TP53 inherited defect, who suffered from bilateral cancer and had ER+/PgR+/HER2− receptor phenotype in both tumors (Supplementary Table S5). Recent studies provided evidence on BC-predisposing role of BRIP1, RAD51C, FANCC and MRE11A germ-line mutations (Supplementary Table S2). Based on established significance of these genes for hereditary BC, we included them into the testing panel. However, no truncating mutations have been identified. We further focused on genes with an unproven role in BC predisposition, which participate in key components of DNA repair (Supplementary Table S2). This effort led to the identification of a recurrent BRCA1-associated mutation in the BLM gene, which has been described in detail in our earlier report [10]. The analysis of a similar gene, WRN, also revealed an instance of heterozygous truncating mutation (R1406X); however, in contrast to BLM, the fulllength sequence examination of the extended series of genetically enriched BC failed to detect additional WRN-heterozygous patients. Other candidate genes, including BARD1, BRD7, CHEK1, DDB2, ERCC1, EXO1, FANCG, PARP1, PARP2, RAD51, RNF8, did not contain protein-truncating mutations in the analyzed BC set. In addition, a number of probably damaging missense mutations have been identified; however none of them turned out to be recurrent (Table 1). Discussion The results of this study may have some implications for breast cancer research and management. In particular, this report confirmed a noticeable occurrence of PALB2 mutations in Slavic BC patients. Given convincing evidence for potential clinical utility of PALB2 testing [19], the status of this gene has to be considered upon routine hereditary BC diagnostic activities. Also, we confirmed nonrandom occurrence of TP53 germ-line mutations in BC patients with clinical features of familial disease predisposition. It remains to be investigated why some TP53 mutation carriers experience multiple primary neoplasms starting from adolescence, while others remain cancer-free at least until midlife and eventually develop single-site breast cancer disease. Interestingly, our study failed to identify novel lesions in well-known BC-predisposing genes, CHEK2 and NBN/NBS1, although recent multigene sequencing efforts revealed that the spectrum of mutations in these genes is not limited to founder alleles [20]. Apart from previously published data on BCpredisposing significance of the BLM Q548X allele [10], we were able
Table 1 Germ-line mutations in DNA repair genes with proven or potential breast cancer predisposing role. Gene
Number of samples analyzed
Truncating or splice site mutations
TP53 TP53 PALB2 PALB2 PALB2 PALB2 BRIP1 BLM BLM EXO1 FANCC NBN/NBS1 NBN/NBS1 PARP1 PARP1 WRN WRN
203 203 165 165 165 165 95 190 95 95 95 95 95 95 95 190 190
c.560-2A>G (n = 1)
Missense variants with presumably pathogenic role
Polyphen-2 prediction
R282W (n = 1)
Probably damaging
p.R173C (n = 1)
Probably damaging
p.R1139P (n = 1) p.N279S (n = 1) p.S26F (n = 1) p.I171V (n = 1) p.R215W (n = 1) p.Q150H (n = 1) p.R857Q (n = 1)
Possibly damaging Probably damaging Probably damaging Probably damaging Probably damaging Probably damaging Probably damaging
p.D143H (n = 1)
Probably damaging
R414X (n = 1) Q921X (n = 1) c.1592delT (n = 1) c.1317delG (n = 1) Q548X (n = 2)
R1406X (n = 1)
References http://p53.iarc.fr/TP53GeneVariations.aspx rs28934574; http://p53.iarc.fr/TP53GeneVariations.aspx [11,13] [11] [14] [12] rs4988345 [10,15] This study rs4149909 [16] [17,18] [18] rs142376976 rs190105316 rs11574410 This study
Please cite this article in press as: Anna P. Sokolenko, et al., Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.01.022
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to identify only 1 sample with truncating mutation, WRN R1406X. Given strong functional similarity between BLM and WRN, the latter gene has to be included in the hereditary BC research agenda, although we could not demonstrate additional instances of the WRN involvement. Interestingly, a number of case–control studies provided evidence for an association between WRN gene polymorphisms and breast cancer risk [21–24]. While the mutations leading to frame-shifts, premature stopcodons or alterations in the splice sites are always considered in candidate hereditary cancer gene studies, the evaluation of significance of missense variants remains significantly more complicated. It is self-explanatory that while truncating mutations of the same protein can usually be pooled together and analyzed as a whole, each amino acid substitution needs to be considered on an individual basis. The existing tools for mutation analysis, such as in silico prediction of potential influence on protein structure, tracking of segregation with the disease in extended pedigrees, comparison of frequencies in cases vs. controls, or direct functional studies with allelic protein variants, are not sufficiently robust and fail to provide conclusive information in most of instances. Rapid accumulation of data obtained by massive parallel sequencing will lead to a dramatic increase in the number of missense germ-line variants with unknown clinical significance. The development of approaches allowing reliable discrimination between disease-causing and relatively neutral amino acid substitutions appears to be a major unmet need of contemporary medical genetic research. Acknowledgements This work was supported by the Russian Scientific Fund (grant number 14-25-00111). We cordially thank Prof. William R. Miller for stimulating discussions. Conflict of interest There are no conflicts of interest in the studies reported in the paper. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.01.022. References [1] U.O. Njiaju, O.I. Olopade, Genetic determinants of breast cancer risk: a review of current literature and issues pertaining to clinical application, Breast J. 18 (2012) 436–442. [2] P. Apostolou, F. Fostira, Hereditary breast cancer: the era of new susceptibility genes, Biomed Res Int. (2013) 747318. [3] N. Bogdanova, S. Helbig, T. Dörk, Hereditary breast cancer: ever more pieces to the polygenic puzzle, Hered Cancer Clin Pract. 11 (2013) 12. [4] F.J. Couch, K.L. Nathanson, K. Offit, Two decades after BRCA: setting paradigms in personalized cancer care and prevention, Science 343 (2014) 1466–1470.
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Please cite this article in press as: Anna P. Sokolenko, et al., Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.01.022