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Journal of Clinical Oncology, Vol 26, No 25 (September 1), 2008: pp. 4212-4214 © 2008 American Society of Clinical Oncology. DOI: 10.1200/JCO.2008.18.2089
Clinical Classification of BRCA1 DNA Missense Variants: H1686Q Is a Novel Pathogenic Mutation Occurring in the Ontogenetically Invariant THV Motif of the N-Terminal BRCT DomainDepartment of Experimental Medicine, University La Sapienza, Rome, Italy
Department of Experimental Medicine, University La Sapienza; Medical Oncology, IDI Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
Department of Experimental Medicine, University La Sapienza, Rome, Italy
Division of Pathology, S. Giovanni Hospital, Torino, Italy
Department of Experimental Medicine, University La Sapienza, Rome, Italy
Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy
Neuromed Institute, Pozzilli, Italy
Department of Experimental Medicine, University La Sapienza, Rome, Italy
Medical Oncology, IDI Istituto di Ricovero e Cura a Carattere Scientifico; Department of Medical Oncology, S. Andrea Hospital, Rome, Italy
Department of Experimental Medicine, University La Sapienza, Rome, Italy
Department of Experimental Medicine, University La Sapienza, Rome; Neuromed Institute, Pozzilli, Italy
Department of Experimental Medicine, University La Sapienza, Rome, Italy
Department of Experimental Medicine, University La Sapienza, Rome; Neuromed Institute, Pozzilli, Italy To the Editor: Genetic testing for BRCA1 and BRCA2 gene mutations is increasingly contributing to the medical management of people at increased risk for breast and/or ovarian cancer.1,2 However, BRCA1/2 alterations readily identifiable as deleterious (such as protein-truncating mutations or genomic rearrangements) can be found in only 20% to 30% of breast cancer families and approximately 50% of breast and ovarian cancer families.3-5 Other genes (such as p53, CHK2, ATM, Brip1, and Palb2) have been linked to inherited breast and ovarian cancer, but their contribution is likely to be modest in statistical terms.6-8 As a consequence, a large number of breast/ovarian cancer families end their path through genetic counseling and testing with uninformative results because of the absence of deleterious mutations in the tested genes or, in a rather large number (approximately 10% to 20%) of cases, because of the identification of unclassified BRCA1/2 variants (ie, missense mutations that cannot be readily classified as pathogenic or nonpathogenic). Indeed, 94% of the 575 missense mutations listed in the BRCA1 Breast Cancer Information Core database (http://research.nhgri.nih.gov/bic/) have an unknown clinical significance, whereas only 6% have already been defined as either clinically relevant or unrelevant. Therefore, definition of the clinical impact of BRCA1/2 unclassified variants is becoming a compelling question for proper risk calculation and clinical management of individuals potentially exposed to a genetic risk for breast/ovarian cancer. We read with great interest a recent article by Malacrida et al9 that demonstrated that the BRCA1 Val1688del is a deleterious mutation that characterizes 1.5% of the breast and ovarian cancer families from northeast Italy. This mutation consists of an in-frame deletion of a valine residue occurring in the first BRCA1 BRCT domain. V1688 falls within a region strikingly conserved among species (Fig 1A). Indeed, this region is involved in binding the phosphoepitope of important BRCA1 interactors such as CtIP and BACH1.10,11 Other mutations occurring within or close to this region may also prove to be pathogenic.
Here, we report on the occurrence of the BRCA1 H1686Q missense mutation in an Italian breast/ovarian cancer family, provide evidence for its pathogenic significance, and discuss the relevance of these observations in light of the important function of this BRCA1 protein domain. Indeed, we recently examined a 49-year-old woman (proband) with a breast cancer diagnosis (age 48 years, grade 3, estrogen and progesterone receptor negative, and HER-2/neu immunohistochemistry positive) and a strong family history of breast/ovarian cancers (Italian CaGENE score = 85; see pedigree in Fig 1B). On the basis of this preliminary evaluation, the proband was subjected to BRCA1/2 genetic screening for point mutations and genomic rearrangements, as previously described.4,12 We were unable to detect any other BRCA1/2 gene mutation, with the exception of the previously unreported 5177 T>A substitution in BRCA1 exon 17, leading to the H1686Q missense substitution. Several parameters may help establish the significance of novel variants, such as the interspecies conservation of the amino acid at the mutation site, the type of substitution and its impact on the protein structure, analysis of cosegregation with disease, co-occurrence with deleterious mutations in the same gene, the features of the tumors eventually occurring in the variant carriers, or the loss of the wild-type allele with conservation of the variant in the tumor tissue. The possibility of integrating data from such different sources to classify BRCA1/2 variants has been addressed in several recent publications.13-18 Finally, laboratory tests, when available, may help define the impact of the variants directly assaying the protein function.19,20 This is the case for missense mutations occurring in the tandem BRCT domains located in the C terminus of BRCA1, which may impair the ability of this region to promote transcription from a synthetic reporter construct. Therefore, we applied this integrated approach to the BRCA1 H1686Q mutation. H1686 is a residue strikingly conserved from man to plant (see alignment, Fig 1A). Analysis of the impact of the H1686Q substitution on BRCA1 protein function performed by the PolyPhen software (http://genetics.bwh.harvard.edu/pph/) predicted that the mutation is probably damaging with a high score (2.696). The Align GVGD (http://agvgd.iarc.fr/) and Sorting Intolerant from Tolerant (http://blocks.fhcrc.org/sift/SIFT.html) programs provided similar predictions. Importantly, the H1686Q mutation occurred in only one breast/ovarian cancer family of the 168 we screened so far and in none of the controls (unaffected individuals). The healthy brother of the proband did not carry the mutation, whereas the breast cancer–and ovarian cancer–affected aunt (the only available affected relative) was a carrier of the H1686Q substitution. We found loss of heterozygosity (with conservation of the 5177 T>A mutant allele) in the breast tumor tissue of the proband and in both breast and ovarian cancer samples of the affected aunt (Fig 1C). All of these observations support the possibility that the H1686Q variant is a pathogenic mutation. A final validation of this hypothesis comes from an observation by Carvalho et al21 that searching for BRCA1 temperature-sensitive mutants generated and characterized in vitro a construct containing the H1686Q mutation. In their analysis, they clearly showed that this is a loss of function mutation, possibly because of its impaired ability to interact with protein partners such as CtIP. Therefore, by all means, the H1686Q missense mutation is a novel BRCA1 pathogenic mutation. Interestingly, H1686 belongs to a THV motif ontogenically invariant from Arabidopsis thaliana to man, suggesting that this amino acid might be important for BRCA1 structural/functional correlation. Indeed, Karchin et al19 observed that these residues might be involved in the formation of a groove on the N-BRCT surface opposite to the cleft involved in phosphoepitope binding. The observation that the T1685I,22 H1686Q (present report), and V1688del9 variants are pathogenic in humans provides definitive evidence for the structural relevance of this motif within the BRCA1 N-BRCT domain. Interestingly, a few amino acids downstream, another motif, the 1699RTKL, is also strikingly conserved among species, and the missense variants involving R1699 (ie, R1699W and R1699Q) are nonfunctional in vitro and pathogenic in vivo.14,20,23 According to our search and the Breast Cancer Information Core database, no other mutation affecting these motifs have been described. However, should a novel missense mutation be found affecting this motif, it would be likely to be pathogenic. In conclusion, we reported here the characterization of the novel H1686Q BRCA1 pathogenic variant that helped us and might help other oncologists define the proper risk and clinical management of carriers belonging to breast/ovarian cancer families. In addition, we delineated an approach that might be used to establish the clinical significance of single missense variants found in breast/ovarian cancer families and provided support to the hypothesis that the THV motif might represent a structurally and functionally important domain of the BRCA1 N-BRCT domain. Finally, we raised the hypothesis that other domains, such us the 1699RTLK motif, might also be potential targets for yet undescribed pathogenic mutations that would need to be promptly characterized. AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author(s) indicated no potential conflicts of interest.
ACKNOWLEDGMENTS This work was partially supported by grants from the Associazione Italiana per la Ricerca sul Cancro, Ministry of Health, National Research Council, Ministry of University and Research, and Pasteur Institute/ Cenci-Bolognetti Foundation. REFERENCES 1. Gulati AP, Domchek SM: The clinical management of BRCA1 and BRCA2 mutation carriers. Curr Oncol Rep 10:47-53, 2008[CrossRef][Medline] 2. Palma M, Ristori E, Ricevuto E, et al: BRCA1 and BRCA2: The genetic testing and the current management options for mutation carriers. Crit Rev Oncol Hematol 57:1-23, 2006[Medline] 3. Martin AM, Blackwood MA, Antin-Ozerkis D, et al: Germline mutations in BRCA1 and BRCA2 in breast-ovarian families from a breast cancer risk evaluation clinic. J Clin Oncol 19:2247-2253, 2001 4. Buffone A, Capalbo C, Ricevuto E, et al: Prevalence of BRCA1 and BRCA2 genomic rearrangements in a cohort of consecutive Italian breast and/or ovarian cancer families. Breast Cancer Res Treat 106:289-296, 2007[CrossRef][Medline] 5. Capalbo C, Buffone A, Vestri A, et al: Does the search for large genomic rearrangements impact BRCAPRO carrier prediction? J Clin Oncol 25:2632-2634, 2007 6. Walsh T, Casadei S, Coats KH, et al: Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer. JAMA 295:1379-1388, 2006 7. Seal S, Thompson D, Renwick A, et al: Truncating mutations in the Fanconi anemia J gene BRIP1 are low-penetrance breast cancer susceptibility alleles. Nat Genet 38:1239-1241, 2006[CrossRef][Medline] 8. Erkko H, Xia B, Nikkila J, et al: A recurrent mutation in PALB2 in Finnish cancer families. Nature 446:316-319, 2007[CrossRef][Medline] 9. Malacrida S, Agata S, Callegaro M, et al: BRCA1 p.Val1688del is a deleterious mutation that recurs in breast and ovarian cancer families from Northeast Italy. J Clin Oncol 26:26-31, 2008 10. Varma AK, Brown RS, Birrane G, et al: Structural basis for cell cycle checkpoint control by the BRCA1-CtIP complex. Biochemistry 44:10941-10946, 2005[CrossRef][Medline] 11. Williams RS, Lee MS, Hau DD, et al: Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1. Nat Struct Mol Biol 11:519-525, 2004[CrossRef][Medline] 12. Capalbo C, Ricevuto E, Vestri A, et al: BRCA1 and BRCA2 genetic testing in Italian breast and/or ovarian cancer families: Mutation spectrum and prevalence and analysis of mutation prediction models. Ann Oncol 17:vii34-vii40, 2006 (suppl 7) 13. Abkevich V, Zharkikh A, Deffenbaugh AM, et al: Analysis of missense variation in human BRCA1 in the context of interspecific sequence variation. J Med Genet 41:492-507, 2004 14. Goldgar DE, Easton DF, Deffenbaugh AM, et al: Integrated evaluation of DNA sequence variants of unknown clinical significance: Application to BRCA1 and BRCA2. Am J Hum Genet 75:535-544, 2004[CrossRef][Medline] 15. Spurdle AB, Lakhani SR, Healey S, et al: Clinical classification of BRCA1 and BRCA2 DNA sequence variants: The value of cytokeratin profiles and evolutionary analysis—A report from the kConFab Investigators. J Clin Oncol 26:1657-1663, 2008 16. Tavtigian SV, Deffenbaugh AM, Yin L, et al: Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J Med Genet 43:295-305, 2006 17. Easton DF, Deffenbaugh AM, Pruss D, et al: A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes. Am J Hum Genet 81:873-883, 2007[CrossRef][Medline] 18. Phelan CM, Dapic V, Tice B, et al: Classification of BRCA1 missense variants of unknown clinical significance. J Med Genet 42:138-146, 2005 19. Karchin R, Monteiro AN, Tavtigian SV, et al: Functional impact of missense variants in BRCA1 predicted by supervised learning. PLoS Comput Biol 3:e26, 2007[CrossRef][Medline] 20. Vallon-Christersson J, Cayanan C, Haraldsson K, et al: Functional analysis of BRCA1 C-terminal missense mutations identified in breast and ovarian cancer families. Hum Mol Genet 10:353-360, 2001 21. Carvalho MA, Billack B, Chan E, et al: Mutations in the BRCT domain confer temperature sensitivity to BRCA1 in transcription activation. Cancer Biol Ther 1:502-508, 2002[Medline] 22. Carvalho MA, Marsillac SM, Karchin R, et al: Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis. Cancer Res 67:1494-1501, 2007 23. Worley T, Vallon-Christersson J, Billack B, et al: A naturally occurring allele of BRCA1 coding for a temperature-sensitive mutant protein. Cancer Biol Ther 1:497-501, 2002[Medline]
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Copyright © 2008 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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