Genetic testing has the potential to guide the prevention and treatment of disease in a variety of settings, and recent technical advances have greatly increased our ability to acquire large amounts of genetic data. The interpretation of this data remains challenging, as the clinical significance of genetic variation detected in the laboratory is not always clear. Although regulatory agencies and professional societies provide some guidance regarding the classification, reporting, and long-term follow-up of variants, few protocols for the implementation of these guidelines have been described. Because the primary aim of clinical testing is to provide results to inform medical management, a variant classification program that offers timely, accurate, confident and cost-effective interpretation of variants should be an integral component of the laboratory process. Here we describe the components of our laboratory's current variant classification program (VCP), based on 20 years of experience and over one million samples tested, using the BRCA1/2 genes as a model. Our VCP has lowered the percentage of tests in which one or more BRCA1/2 variants of uncertain significance (VUSs) are detected to 2.1% in the absence of a pathogenic mutation, demonstrating how the coordinated application of resources toward classification and reclassification significantly impacts the clinical utility of testing.

BACKGROUND: Current estimates of the contribution of large rearrangement (LR) mutations in the BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 2, early onset) genes responsible for hereditary breast and ovarian cancer are based on limited studies of relatively homogeneous patient populations. The prevalence of BRCA1/2 LRs was investigated in 48,456 patients with diverse clinical histories and ancestries, referred for clinical molecular testing for suspicion of hereditary breast and ovarian cancer. METHODS: Sanger sequencing analysis was performed for BRCA1/2 and LR testing for deletions and duplications using a quantitative multiplex polymerase chain reaction assay. Prevalence data were analyzed for patients from different risk and ethnic groups between July 2007 and April 2011. Patients were designated as “high-risk” if their clinical history predicted a high prior probability, wherein LR testing was performed automatically in conjunction with sequencing. “Elective” patients did not meet the high-risk criteria, but underwent LR testing as ordered by the referring health care provider. RESULTS: Overall BRCA1/2 mutation prevalence among high-risk patients was 23.8% versus 8.2% for the elective group. The mutation profile for high-risk patients was 90.1% sequencing mutations versus 9.9% LRs, and for elective patients, 94.1% sequencing versus 5.9% LRs. This difference may reflect the bias in high-risk patients to carry mutations in BRCA1, which has a higher penetrance and frequency of LRs compared with BRCA2. There were significant differences in the prevalence and types of LRs in patients of different ancestries. LR mutations were significantly more common in Latin American/Caribbean patients. CONCLUSIONS: Comprehensive LR testing in conjunction with full gene sequencing is an appropriate strategy for clinical BRCA1/2 analysis.

An estimated 1:40 individuals of Ashkenazi Jewish (AJ) ancestry carry one of three common founder mutations in BRCA1 or BRCA2, resulting in the inherited cancer condition, Hereditary Breast and Ovarian Cancer (HBOC) syndrome. Targeted testing for these three mutations (BRCA1 187delAG, BRCA1 5385insC, and BRCA2 6174delT) is therefore recommended for all AJ breast and ovarian cancer patients, regardless of age of diagnosis or family history. Comprehensive analysis of both genes is recommended for a subset of AJ patients in whom founder mutations are not identified, but estimates of the yield from comprehensive analysis in this population vary widely. We sought to determine the proportion of non-founder mutations as a percentage of all mutations in BRCA1 and BRCA2 among AJ patients to inform decisions about HBOC testing strategies in this population. We analyzed the genetic testing results for 37,952 AJ patients for whom clinical testing of BRCA1 and BRCA2 was performed at Myriad Genetic Laboratories from January 2006 through August 2013. Analysis was limited to AJ-only patients for whom the initial test order was either (1) comprehensive testing, or (2) founder mutation testing with instructions to automatically "reflex" to comprehensive analysis if negative. Cases were excluded if a separate follow-up order was placed to reflex to comprehensive analysis only after the founder mutation testing was reported out as negative. Among all BRCA1 and BRCA2 mutations detected in these groups, the percentage of non-founder mutations was 13 % (104/802) and 7.2 % (198/2,769). One-hundred and eighty-nine unique non-founder mutations were detected, 76 in BRCA1 and 113 in BRCA2. Non-founder mutations make up between 7.2 and 13.0 % of all BRCA1 and BRCA2 mutations in Ashkenazi Jews. A wide range of mutations are present, most of which are also seen in non-AJ individuals.

These findings demonstrate that a GC-mandate policy implemented by a LNHP substantially decreased access to appropriate genetic testing, disproportionately impacting minority populations without any evidence that inappropriate testing was decreased.