Analysis of Italian BRCA1/2 Pathogenic Variants Identifies a Private Spectrum in the Population from the Bergamo Province in Northern Italy
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Distribution of the PVs Found in BGP
2.2. Comparison between the BGP and Non-BGP PV Spectra
3. Discussion
4. Materials and Methods
4.1. BRCA1/2 PV Carriers
4.2. Heterogeneity of the BRCA1/2 PV Carriers
4.3. Statistical Analyses
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Kuchenbaecker, K.B.; Hopper, J.L.; Barnes, D.R.; Phillips, K.A.; Mooij, T.M.; Roos-Blom, M.J.; Jervis, S.; van Leeuwen, F.E.; Milne, R.L.; Andrieu, N.; et al. Risks of Breast, Ovarian, and Contralateral Breast Cancer for BRCA1 and BRCA2 Mutation Carriers. JAMA 2017, 317, 2402–2416. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fackenthal, J.D.; Olopade, O.I. Breast cancer risk associated with BRCA1 and BRCA2 in diverse populations. Nat. Rev. Cancer 2007, 7, 937–948. [Google Scholar] [CrossRef] [PubMed]
- Janavicius, R. Founder BRCA1/2 mutations in the Europe: Implications for hereditary breast-ovarian cancer prevention and control. EPMA J. 2010, 1, 397–412. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Turnbull, C.; Rahman, N. Genetic predisposition to breast cancer: Past, present, and future. Annu. Rev. Genomics Hum. Genet. 2008, 9, 321–345. [Google Scholar] [CrossRef] [PubMed]
- Roa, B.B.; Boyd, A.A.; Volcik, K.; Richards, C.S. Ashkenazi Jewish population frequencies for common mutations in BRCA1 and BRCA2. Nat. Genet. 1996, 14, 185–187. [Google Scholar] [CrossRef]
- Tonin, P.; Weber, B.; Offit, K.; Couch, F.; Rebbeck, T.R.; Neuhausen, S.; Godwin, A.K.; Daly, M.; Wagner-Costalos, J.; Berman, D.; et al. Frequency of recurrent BRCA1 and BRCA2 mutations in Ashkenazi Jewish breast cancer families. Nat. Med. 1996, 2, 1179–1183. [Google Scholar] [CrossRef]
- Johannesdottir, G.; Gudmundsson, J.; Bergthorsson, J.T.; Arason, A.; Agnarsson, B.A.; Eiriksdottir, G.; Johannsson, O.T.; Borg, A.; Ingvarsson, S.; Easton, D.F.; et al. High prevalence of the 999del5 mutation in icelandic breast and ovarian cancer patients. Cancer Res. 1996, 56, 3663–3665. [Google Scholar]
- Thorlacius, S.; Sigurdsson, S.; Bjarnadottir, H.; Olafsdottir, G.; Jonasson, J.G.; Tryggvadottir, L.; Tulinius, H.; Eyfjord, J.E. Study of a single BRCA2 mutation with high carrier frequency in a small population. Am. J. Hum. Genet. 1997, 60, 1079–1084. [Google Scholar]
- Sarantaus, L.; Huusko, P.; Eerola, H.; Launonen, V.; Vehmanen, P.; Rapakko, K.; Gillanders, E.; Syrjakoski, K.; Kainu, T.; Vahteristo, P.; et al. Multiple founder effects and geographical clustering of BRCA1 and BRCA2 families in Finland. Eur. J. Hum. Genet. 2000, 8, 757–763. [Google Scholar] [CrossRef]
- Hatzikotoulas, K.; Gilly, A.; Zeggini, E. Using population isolates in genetic association studies. Brief. Funct. Genomics 2014, 13, 371–377. [Google Scholar] [CrossRef] [Green Version]
- Xue, Y.; Mezzavilla, M.; Haber, M.; McCarthy, S.; Chen, Y.; Narasimhan, V.; Gilly, A.; Ayub, Q.; Colonna, V.; Southam, L.; et al. Enrichment of low-frequency functional variants revealed by whole-genome sequencing of multiple isolated European populations. Nat. Commun. 2017, 8, 15927. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zeggini, E. Using genetically isolated populations to understand the genomic basis of disease. Genome Med. 2014, 6, 83. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Cocca, M.; Barbieri, C.; Concas, M.P.; Robino, A.; Brumat, M.; Gandin, I.; Trudu, M.; Sala, C.F.; Vuckovic, D.; Girotto, G.; et al. A bird’s-eye view of Italian genomic variation through whole-genome sequencing. Eur. J. Hum. Genet. 2020, 28, 435–444. [Google Scholar] [CrossRef] [PubMed]
- Colonna, V.; Pistis, G.; Bomba, L.; Mona, S.; Matullo, G.; Boano, R.; Sala, C.; Vigano, F.; Torroni, A.; Achilli, A.; et al. Small effective population size and genetic homogeneity in the Val Borbera isolate. Eur. J. Hum. Genet. 2013, 21, 89–94. [Google Scholar] [CrossRef] [Green Version]
- Fiorito, G.; Di Gaetano, C.; Guarrera, S.; Rosa, F.; Feldman, M.W.; Piazza, A.; Matullo, G. The Italian genome reflects the history of Europe and the Mediterranean basin. Eur. J. Hum. Genet. 2016, 24, 1056–1062. [Google Scholar] [CrossRef] [Green Version]
- Grugni, V.; Raveane, A.; Mattioli, F.; Battaglia, V.; Sala, C.; Toniolo, D.; Ferretti, L.; Gardella, R.; Achilli, A.; Olivieri, A.; et al. Reconstructing the genetic history of Italians: New insights from a male (Y-chromosome) perspective. Ann. Hum. Biol. 2018, 45, 44–56. [Google Scholar] [CrossRef]
- Raveane, A.; Aneli, S.; Montinaro, F.; Athanasiadis, G.; Barlera, S.; Birolo, G.; Boncoraglio, G.; Di Blasio, A.M.; Di Gaetano, C.; Pagani, L.; et al. Population structure of modern-day Italians reveals patterns of ancient and archaic ancestries in Southern Europe. Sci. Adv. 2019, 5, eaaw3492. [Google Scholar] [CrossRef] [Green Version]
- Sazzini, M.; Gnecchi Ruscone, G.A.; Giuliani, C.; Sarno, S.; Quagliariello, A.; De Fanti, S.; Boattini, A.; Gentilini, D.; Fiorito, G.; Catanoso, M.; et al. Complex interplay between neutral and adaptive evolution shaped differential genomic background and disease susceptibility along the Italian peninsula. Sci. Rep. 2016, 6, 32513. [Google Scholar] [CrossRef] [Green Version]
- Baudi, F.; Quaresima, B.; Grandinetti, C.; Cuda, G.; Faniello, C.; Tassone, P.; Barbieri, V.; Bisegna, R.; Ricevuto, E.; Conforti, S.; et al. Evidence of a founder mutation of BRCA1 in a highly homogeneous population from southern Italy with breast/ovarian cancer. Hum. Mutat. 2001, 18, 163–164. [Google Scholar] [CrossRef]
- Pisano, M.; Cossu, A.; Persico, I.; Palmieri, G.; Angius, A.; Casu, G.; Palomba, G.; Sarobba, M.G.; Rocca, P.C.; Dedola, M.F.; et al. Identification of a founder BRCA2 mutation in Sardinia. Br. J. Cancer 2000, 82, 553–559. [Google Scholar] [CrossRef] [Green Version]
- Caligo, M.A.; Ghimenti, C.; Cipollini, G.; Ricci, S.; Brunetti, I.; Marchetti, V.; Olsen, R.; Neuhausen, S.; Shattuck-Eidens, D.; Conte, P.F.; et al. BRCA1 germline mutational spectrum in Italian families from Tuscany: A high frequency of novel mutations. Oncogene 1996, 13, 1483–1488. [Google Scholar] [PubMed]
- Papi, L.; Putignano, A.L.; Congregati, C.; Zanna, I.; Sera, F.; Morrone, D.; Falchetti, M.; Turco, M.R.; Ottini, L.; Palli, D.; et al. Founder mutations account for the majority of BRCA1-attributable hereditary breast/ovarian cancer cases in a population from Tuscany, Central Italy. Breast Cancer Res. Treat. 2009, 117, 497–504. [Google Scholar] [CrossRef] [PubMed]
- Malacrida, S.; Agata, S.; Callegaro, M.; Casella, C.; Barana, D.; Scaini, M.C.; Manoukian, S.; Oliani, C.; Radice, P.; Barile, M.; et al. BRCA1 p.Val1688del is a deleterious mutation that recurs in breast and ovarian cancer families from Northeast Italy. J. Clin. Oncol. 2008, 26, 26–31. [Google Scholar] [CrossRef] [PubMed]
- Cini, G.; Mezzavilla, M.; Della Puppa, L.; Cupelli, E.; Fornasin, A.; D’Elia, A.V.; Dolcetti, R.; Damante, G.; Bertok, S.; Miolo, G.; et al. Tracking of the origin of recurrent mutations of the BRCA1 and BRCA2 genes in the North-East of Italy and improved mutation analysis strategy. BMC Med. Genet. 2016, 17, 11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Caleca, L.; Putignano, A.L.; Colombo, M.; Congregati, C.; Sarkar, M.; Magliery, T.J.; Ripamonti, C.B.; Foglia, C.; Peissel, B.; Zaffaroni, D.; et al. Characterization of an Italian founder mutation in the RING-finger domain of BRCA1. PLoS ONE 2014, 9, e86924. [Google Scholar] [CrossRef] [PubMed]
- Catucci, I.; Casadei, S.; Ding, Y.C.; Volorio, S.; Ficarazzi, F.; Falanga, A.; Marchetti, M.; Tondini, C.; Franchi, M.; Adamson, A.; et al. Haplotype analyses of the c.1027C>T and c.2167_2168delAT recurrent truncating mutations in the breast cancer-predisposing gene PALB2. Breast Cancer Res. Treat. 2016, 160, 121–129. [Google Scholar] [CrossRef] [Green Version]
- Catucci, I.; Peterlongo, P.; Ciceri, S.; Colombo, M.; Pasquini, G.; Barile, M.; Bonanni, B.; Verderio, P.; Pizzamiglio, S.; Foglia, C.; et al. PALB2 sequencing in Italian familial breast cancer cases reveals a high-risk mutation recurrent in the province of Bergamo. Genet. Med. 2014, 16, 688–694. [Google Scholar] [CrossRef] [Green Version]
- Laitman, Y.; Friebel, T.M.; Yannoukakos, D.; Fostira, F.; Konstantopoulou, I.; Figlioli, G.; Bonanni, B.; Manoukian, S.; Zuradelli, M.; Tondini, C.; et al. The spectrum of BRCA1 and BRCA2 pathogenic sequence variants in Middle Eastern, North African, and South European countries. Hum. Mutat. 2019, 40, e1–e23. [Google Scholar] [CrossRef] [Green Version]
- Rebbeck, T.R.; Friebel, T.M.; Friedman, E.; Hamann, U.; Huo, D.; Kwong, A.; Olah, E.; Olopade, O.I.; Solano, A.R.; Teo, S.H.; et al. Mutational spectrum in a worldwide study of 29,700 families with BRCA1 or BRCA2 mutations. Hum. Mutat. 2018, 39, 593–620. [Google Scholar] [CrossRef] [Green Version]
- Yang, X.; Leslie, G.; Doroszuk, A.; Schneider, S.; Allen, J.; Decker, B.; Dunning, A.M.; Redman, J.; Scarth, J.; Plaskocinska, I.; et al. Cancer Risks Associated With Germline PALB2 Pathogenic Variants: An International Study of 524 Families. J. Clin. Oncol. 2020, 38, 674–685. [Google Scholar] [CrossRef]
- Palmieri, G.; Palomba, G.; Cossu, A.; Pisano, M.; Dedola, M.F.; Sarobba, M.G.; Farris, A.; Olmeo, N.; Contu, A.; Pasca, A.; et al. BRCA1 and BRCA2 germline mutations in Sardinian breast cancer families and their implications for genetic counseling. Ann. Oncol. 2002, 13, 1899–1907. [Google Scholar] [CrossRef] [PubMed]
- De Nicolo, A.; Parisini, E.; Zhong, Q.; Dalla Palma, M.; Stoeckert, K.A.; Domchek, S.M.; Nathanson, K.L.; Caligo, M.A.; Vidal, M.; Cusick, M.E.; et al. Multimodal assessment of protein functional deficiency supports pathogenicity of BRCA1 p.V1688del. Cancer Res. 2009, 69, 7030–7037. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Barnholtz-Sloan, J.S.; Chakraborty, R.; Sellers, T.A.; Schwartz, A.G. Examining population stratification via individual ancestry estimates versus self-reported race. Cancer Epidemiol. Biomark. Prev. 2005, 14, 1545–1551. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Altman, D.G.; Machin, D.; Bryant, T.N.; Gardner, M.J. Statistics with Confidence, 2nd ed.; BMJ Books: Wiley, UK, 2000; p. 49. [Google Scholar]
PVs | Total No. of Carriers | Carrier Distribution (No. of Carriers; Freq%) | Suggested Origin | |
---|---|---|---|---|
BGP | Regions | |||
Common (10) | ||||
BRCA1:c.190T>C | 55 | (34; 34.0) | Molise (1; 100), Trentino Alto Adige (1; 11.1), Veneto (2; 8.3), Friuli Venezia Giulia (1; 7.1), Lombardy non-BGP (14; 4.8), Piedmont (2; 1.2) | BGP 1 |
BRCA2:c.5904_5907delAGTC | 15 | (11; 11.0) | Lombardy non-BGP (4; 1.4) | BGP 1 |
BRCA1:c.2727_2730delTCAA | 15 | (3; 3.0) | Lombardy non-BGP (12; 4.1) | BGP/Lombardy 1 |
BRCA2:c.6469C>T | 7 | (3; 3.0) | Lombardy non-BGP (4; 1.4) | BGP 1 |
BRCA2:c.6625dupA | 4 | (3; 3.0) | Lombardy non-BGP (1; 0.3) | BGP 1 |
BRCA2:c.8878C>T | 34 | (8; 8.0) | Liguria (2; 12.5), Marche (1; 8.3), Lombardy non-BGP (15; 5.1), Veneto (1; 4.2), Piedmont (6; 3.6), Emilia Romagna (1; 2.4) | BGP/Liguria 1 |
BRCA1:c.5062_5064delGTT | 16 | (6; 6.0) | Trentino Alto Adige (2; 22.2), Veneto (2; 8.3), Lombardy non-BGP (5; 1.7), Piedmont (1; 0.6) | BGP/Trentino Alto Adige 1 |
BRCA1:c.3481_3491del11 | 9 | (4; 4.0) | Emilia Romagna (3; 7.3), Lombardy non-BGP (2; 0.7) | BGP/Emilia Romagna 2 |
BRCA1:c.5030_5033delCTAA | 21 | (4; 4.0) | Liguria (3; 18.8), Piedmont (6; 3.6), Sicily (2; 3.2), Emilia Romagna (1; 2.4), Lombardy non-BGP (5; 1.7) | BGP/Liguria 2 |
BRCA2:c.7180A>T | 20 | (4; 4.0) | Friuli Venezia Giulia (1; 7.1), Lombardy non-BGP (12; 4.1), Piedmont (3; 1.8) | BGP/Friuli Venezia Giulia 1 |
Rare (15) | ||||
BRCA1:c.406delA | 3 | (2; 2.0) | Lombardy non-BGP (1; 0.3) | BGP 1 |
BRCA2:c.2748delT | 3 | (2; 2.0) | Lombardy non-BGP (1; 0.3) | BGP 1 |
BRCA2:c.5473dupG | 2 | (2; 2.0) | None | BGP 1 |
BRCA2:c.8085_8086insGG | 2 | (2; 2.0) | None | BGP 1 |
BRCA1:c.53T>C | 1 | (1; 1.0) | None | BGP 1 |
BRCA1:c.1961dupA | 1 | (1; 1.0) | None | BGP 2 |
BRCA1:c.2960dupA | 6 | (1; 1.0) | Lombardy non-BGP (5; 1.7) | BGP/Lombardy 1 |
BRCA1:c.3257T>A | 8 | (1; 1.0) | Lombardy non-BGP (5; 1.7), Piedmont (2; 1.2) | BGP/Lombardy 1 |
BRCA2:c.7921_7926delGAATTTinsAG | 2 | (1; 1.0) | Lombardy non-BGP (1; 0.3) | BGP 1 |
BRCA2:c.7976+1G>A | 2 | (1; 1.0) | Lombardy non-BGP (1; 0.3) | BGP 2 |
BRCA2:c.8021dupA | 1 | (1; 1.0) | None | BGP 1 |
BRCA2:c.6468_6469delTC | 15 | (2; 2.0) | Veneto (2; 8.3), Marche (1; 8.3), Tuscany (1; 4.3), Campania (3; 7.5), Apulia (3; 2.1), Piedmont (3; 1.8) | BGP/Campania 1 |
BRCA2:c.1813delA | 2 | (1; 1.0) | Basilicata (1; 11.1) | BGP/Basilicata 2 |
BRCA1:c.514delC | 15 | (1; 1.0) | Sicily (8; 12.7), Lombardy non-BGP (4; 1.4), Apulia (1; 1.0), Piedmont (1; 0.6) | Sicily 1 |
BRCA1:c.5266dupC | 84 | (1; 1.0) | Apulia (31; 30.1), Abruzzo (2; 15.4), Trentino Alto Adige (1; 11.1), Sicily (5; 7.9), Calabria (3; 7.7), Campania (3; 7.5), Lombardy non-BGP (22; 7.5), Piedmont (12; 7.2), Friuli Venezia Giulia (1; 7.1), Liguria (1; 6.3), Emilia Romagna (2; 4.9) | Apulia 2 |
Non-BGP PV | Non-BGP Carriers (%) | BGP Carriers (%) | BGP—Non-BGP Frequency Difference (95%CI) | p-Value |
---|---|---|---|---|
BRCA1:c.5266dupC | 83 (9.0) | 1 (1.0) | −8 (−10.2 to −3.3) | 0.0056 |
BRCA1:c.190T>C | 21 (2.3) | 34 (34.0) | 31.7 (23.1 to 41.5) | <0.0001 |
BRCA1:c.5062_5064delGTT | 10 (1.1) | 6 (6.0) | 4.9 (1.6 to 11.4) | 0.0002 |
BRCA2:c.8878C>T | 26 (2.8) | 8 (8.0) | 5.2 (1.1 to 12.2) | 0.0063 |
BRCA2:c.7180A>T | 16 (1.7) | 4 (4.0) | 2.3 (−0.4 to 8.1) | 0.1219 |
BRCA1:c.5030_5033delCTAA | 17 (1.8) | 4 (4.0) | 2.2 (−0.5 to 8) | 0.1509 |
BRCA1:c.1088delA | 25 (2.7) | 0 (0.0) | −2.7 (−4 to 1.1) | 0.0951 |
BRCA1:c.181T>G | 20 (2.2) | 0 (0.0) | −2.2 (−3.3 to 1.6) | 0.1361 |
BRCA2:c.5796_5797delTA | 15 (1.6) | 0 (0.0) | −1.6 (−2.7 to 2.1) | 0.1986 |
BRCA1:c.3257T>G | 15 (1.6) | 0 (0.0) | −1.6 (−2.7 to 2.1) | 0.1986 |
Testing-Ascertainment Center | All Carriers | Carriers of BRCA1 PVs (%) | Carriers of BRCA2 PVs (%) |
---|---|---|---|
Cogentech-IEO | 353 | 195 (55.2) | 158 (44.8) |
Cogentech-INT | 309 | 187 (60.5) | 122 (39.5) |
Cogentech-OPG * | 90 | 53 (58.9) | 38 (41.1) |
Cogentech-ICH | 61 | 39 (63.9) | 22 (36.1) |
ACSS * | 206 | 107 (51.9) | 100 (48.1) |
All cohorts | 1019 * | 581 (57.0) | 440 (43.0) |
BGP | 100 | 59 (59.0) | 41 (41.0) |
non-BGP * | 919 | 522 (56.8) | 399 (43.2) |
All | 1019 * | 581 (57.0) | 440 (43.0) |
Region | Region of Birth of Carrier Ascertained in | Number of Residents # | Rate * (1:100,000) | |||
---|---|---|---|---|---|---|
All Cities | Milan (INT, IEO, ICH) | Turin (ACSS) | Bergamo (OPG) | |||
Lombardy (Non-BGP) | 293 | 281 | 3 | 9 | 8,987,585 | 3.3 |
BGP | 100 | 29 | 0 | 71 | 1,116,384 | 9.0 |
Lazio | 20 | 18 | 2 | 0 | 5,865,544 | 0.3 |
Campania | 40 | 32 | 7 | 1 | 5,785,861 | 0.7 |
Sicily | 63 | 50 | 9 | 4 | 4,968,410 | 1.3 |
Veneto | 24 | 19 | 4 | 1 | 4,907,704 | 0.5 |
Emilia Romagna | 41 | 35 | 5 | 1 | 4,467,118 | 0.9 |
Piedmont | 167 | 43 | 124 | 0 | 4,341,375 | 3.9 |
Apulia | 103 | 85 | 17 | 1 | 4,008,296 | 2.6 |
Tuscany | 23 | 22 | 1 | 0 | 3,722,729 | 0.6 |
Calabria | 39 | 27 | 12 | 0 | 1,924,701 | 2.0 |
Sardinia | 22 | 17 | 3 | 2 | 1,630,474 | 1.4 |
Liguria | 16 | 13 | 3 | 0 | 1,543,127 | 1.0 |
Marche | 12 | 11 | 1 | 0 | 1,518,400 | 0.8 |
Abruzzo | 13 | 10 | 3 | 0 | 1,305,770 | 1.0 |
Friuli Venezia Giulia | 14 | 8 | 6 | 0 | 1,211,357 | 1.2 |
Trentino Alto Adige | 9 | 9 | 0 | 0 | 1,074,819 | 0.8 |
Umbria | 3 | 3 | 0 | 0 | 880,285 | 0.3 |
Basilicata | 9 | 6 | 3 | 0 | 556,934 | 1.6 |
Molise | 1 | 1 | 0 | 0 | 302,265 | 0.3 |
Aosta Valley | 7 | 4 | 3 | 0 | 125,501 | 5.6 |
All regions (non-BGP) | 919 | 694 | 206 | 19 | 59,128,255 | 1.6 |
All regions | 1019 | 723 | 206 | 90 | 60,244,639 | 1.7 |
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Figlioli, G.; De Nicolo, A.; Catucci, I.; Manoukian, S.; Peissel, B.; Azzollini, J.; Beltrami, B.; Bonanni, B.; Calvello, M.; Bondavalli, D.; et al. Analysis of Italian BRCA1/2 Pathogenic Variants Identifies a Private Spectrum in the Population from the Bergamo Province in Northern Italy. Cancers 2021, 13, 532. https://doi.org/10.3390/cancers13030532
Figlioli G, De Nicolo A, Catucci I, Manoukian S, Peissel B, Azzollini J, Beltrami B, Bonanni B, Calvello M, Bondavalli D, et al. Analysis of Italian BRCA1/2 Pathogenic Variants Identifies a Private Spectrum in the Population from the Bergamo Province in Northern Italy. Cancers. 2021; 13(3):532. https://doi.org/10.3390/cancers13030532
Chicago/Turabian StyleFiglioli, Gisella, Arcangela De Nicolo, Irene Catucci, Siranoush Manoukian, Bernard Peissel, Jacopo Azzollini, Benedetta Beltrami, Bernardo Bonanni, Mariarosaria Calvello, Davide Bondavalli, and et al. 2021. "Analysis of Italian BRCA1/2 Pathogenic Variants Identifies a Private Spectrum in the Population from the Bergamo Province in Northern Italy" Cancers 13, no. 3: 532. https://doi.org/10.3390/cancers13030532