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Journal of Clinical Oncology, Vol 26, No 1 (January 1), 2008: pp. 16-17 © 2008 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.14.4154
Genetic Variants of Uncertain Significance: Flies in the OintmentAbramson Cancer Center, University of Pennsylvania, Philadelphia; and GlaxoSmithKline, King of Prussia, PA Genetic testing for BRCA1 and BRCA2 mutations in individuals with a personal and family history of breast or ovarian cancer can be an invaluable tool in determining appropriate clinical management. Women with such mutations are at high risk for the development of breast and ovarian cancer, and strategies exist to reduce these risks substantially.1 Cancer risk models may be used to help determine appropriate candidates for genetic testing, which we recommend for those with greater than a 5% to 10% probability of carrying a mutation. Positive results from genetic testing for BRCA1 and BRCA2 mutations, although difficult for patients and families to hear, provide both a basis for decision making and, for many people, a measure of peace that comes from answering the question, "Why?" Negative results in members of families with known mutations provide tremendous relief, as well as freedom from intensive surveillance and surgery. Negative results, when a family mutation has not been identified, may be frustrating but do suggest a reduced risk of ovarian cancer, because most heritable susceptibility to ovarian cancer is a result of BRCA1 and BRCA2 mutations. However, undergoing genetic testing and being presented with a variant of uncertain significance (VUSs) is a clinically and emotionally difficult situation. Advising individuals with a VUS on surveillance and prophylactic surgery is challenging for providers, confusing to families, and distressing for everyone. VUSs (also termed unclassified variants) are sequence variations in a gene where the effect of the sequence change on the function of the protein is not known. Such sequence variants in the coding regions of BRCA1 or BRCA2 may or may not be associated with an increased risk of breast, ovarian, or other cancers. Many of these variants are single-nucleotide substitutions (also called missense mutations) that result in a single amino acid change. Some, like the one described in this issue of the Journal of Clinical Oncology by Malacrida et al,2 are in-frame deletions that remove amino acids in multiples of three, and thus do not induce a frame shift in protein translation. Some missense mutations clearly alter the function of BRCA1, such as those that occur in the "ring finger " domain or induce frame shifts by altering splice sites. However, the functional significance of most of the thousands of missense mutations identified to date in BRCA1 and BRCA2 is not clear. Most VUSs are benign polymorphisms, but determining the functional and thus clinical significance of a specific VUS with certainty is difficult. VUSs are unfortunately a common problem in all types of genetic testing, but are particularly problematic in testing BRCA1 and BRCA2 because of the large size of these genes. Between 10% and 15% of individuals undergoing genetic testing for BRCA1 and BRCA2 mutations will be found to have a VUS.3 VUSs are even more common in non-white populations, with frequencies as high as 46% in African Americans4 and 22% in Hispanics.5 The increased difficulty in categorizing VUSs in these populations occurs because the smaller number of people tested to date limits many of the approaches to classifying VUSs described later herein. Fortunately, there are a number of factors which, if known, provide considerable confidence in determining the clinical significance of a VUS (Table 1). Particularly when taken together, these considerations can be important tools when counseling individuals with a VUS.6-9 First, and whenever possible, it is important to determine whether the VUS segregates with breast and ovarian cancer in the family under consideration and in other families with the same VUS. Doing so requires the availability and consent of multiple other family members, including at least one individual with breast or ovarian cancer, to be tested for the VUS. Second, the phenotype of both the family and the tumors should be examined. For example, in the study by Malacrida et al, families with early onset and bilateral breast cancers, multiple women with both breast and ovarian cancer, and "BRCA1-like" breast cancers (medullary and "triple negative" breast cancers that are estrogen-receptor, progesterone-receptor, and ERBB2 negative) provided a strong clinical argument that a BRCA1 mutation was present. The co-segregation of BRCA1 p.Val1688del with cancer in two families—all affected family members carried the VUS, whereas multiple unaffected women did not—provided additional evidence that BRCA1 p.Val1688del is a disease-associated mutation. However, even with that evidence, the possibility existed that BRCA1 p.Val1688del was co-segregating with the "real," undetected, BRCA1 mutation. Thus, additional supporting data were required.
Additional support for BRCA1 p.Val1688del as a deleterious mutation comes from epidemiologic data. If the prevalence of a VUS is the same in individuals with cancer and an unaffected control population, the VUS is unlikely to be associated with substantial cancer risk. In addition, multiple lines of evidence suggest that inheriting two functionally inactive copies of BRCA1 is a lethal event in embryonic development. Therefore, when a VUS is found in one copy of BRCA1 and a known deleterious mutation is found in the other (in trans), the VUS is unlikely to be deleterious. Similarly, two deleterious mutations in the same copy of a gene (in cis) are rare. Malacrida et al did not identify BRCA1 p.Val1688del in an unaffected control population, and it has not been seen in conjunction with other deleterious mutations, providing further support for clinical relevance. Evolutionary data also help. Sequence information for BRCA1 and BRCA2 is available for many species and identifies regions of gene sequence that are highly conserved between species. Allelic variation in these regions, particularly when the variation results in a nonconservative amino acid change, increases the likelihood that the variant is functionally significant. BRCA1 p.Val1688del falls within the BRCT domain, a highly conserved region essential for normal DNA damage repair, providing more evidence that it is deleterious. Loss of heterozygosity in tumors from VUS carriers, with loss of the wild-type allele of BRCA1 and retention of the variant allele, provides yet more evidence for functional significance and was seen in both of the tumor specimens examined by Malacrida et al. When all this genetic evidence is available, a VUS may be categorized with a high degree of confidence. The final step in analyzing a VUS, in vitro proof of loss of BRCA1 function, requires assays to measure the ability of the mutant protein to maintain genomic stability and modulate error-free, homologous, recombination-mediated DNA repair. Even these complex, time-consuming in vitro assays may not fully capture the complicated in vivo functions of BRCA1 and BRCA2, and are performed accurately in only a handful of academic research laboratories in the world. Thus, functional evidence is rarely available for VUS categorization and is not a feasible expectation considering the thousands of BRCA1 and BRCA2 VUSs that have been reported to date. In summary, the characterization of a VUS is difficult work. Unfortunately, most VUSs have been observed only in single families and the detailed analyses as described herein often are not possible. In clinical practice, small family size and affected individuals who are deceased may limit the ability of clinicians to examine co-segregation, and assays for loss of heterozygosity and in vitro function are not readily available. Given that reality, structure-function models that consider evolutionary conservation and the posterior probability of having a mutation based on family and tumor characteristics are likely to be more widely applicable. Yet, despite the challenges, the problem of VUSs is one worth tackling, because the ability to confidently classify a VUS as disease associated or benign has profound clinical significance for families with these variants. As uptake of genetic testing services increases, more and more individuals will be found to have a VUS. In addition, as demonstrated by Malacrida et al, some of these VUSs are common and, if shown to be deleterious, can account for a substantial portion of mutations in selected populations. The demonstration that BRCA1 p.Val1688del is not only deleterious but also a founder mutation that accounts for more BRCA1-associated breast cancer in Italian families than any other single mutation is convincing and important, and is a great service to all those families that have been and will be tested for this mutation. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author(s) indicated no potential conflicts of interest. AUTHOR CONTRIBUTIONS Conception and design: Susan Domchek, Barbara L. Weber Data analysis and interpretation: Susan Domchek, Barbara L. Weber Manuscript writing: Susan Domchek, Barbara L. Weber Final approval of manuscript: Barbara L. Weber
REFERENCES 1. Domchek SM, Weber BL. Clinical management of BRCA1 and BRCA2 mutation carriers. Oncogene 25:5825-5831, 2006[CrossRef][Medline] 2. Malacrida S, Agata S, Callegara M, et al: BRCA1 p.Val1688del is a deleterious mutation that recurs in breast and ovarian cancer families from Northeast Italy. J Clin Oncol 26:10.1200/JCO.2007.13.2118 3. Frank TS, Deffenbaugh AM, Reid JE, et al: Clinical characteristics of individuals with germline mutations in BRCA1 and BRCA2: Analysis of 10,000 individuals. J Clin Oncol 20:1480-1490, 2002 4. Nanda R, Schumm LP, Cummings S, et al: Genetic testing in an ethnically diverse cohort of high-risk women: A comparative analysis of BRCA1 and BRCA2 mutations in American families of European and African ancestry. JAMA 294:1925-1933, 2005 5. Weitzel JN, Lagos V, Blazer KR, et al: Prevalence of BRCA mutations and founder effect in high-risk Hispanic families. Cancer Epidemiol Biomarkers Prev 14:1666-1671, 2005 6. Wu K, Hinson SR, Ohashi A, Farrugia et al: Functional evaluation and cancer risk assessment of BRCA2 unclassified variants. Cancer Res 65:417-426, 2005 7. Chenevix-Trench G, Healey S, Lakhani S, et al: Genetic and histopathologic evaluation of BRCA1 and BRCA2 DNA sequence variants of unknown clinical significance. Cancer Res 66:2019-2027, 2006 8. Mirkovic N, Marti-Renom MA, Weber BL, et al: Structure-based assessment of missense mutations in human BRCA1: Implications for breast and ovarian cancer predisposition. Cancer Res 64:3790-3797, 2004 9. 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] Related Article
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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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