Solving Missing Heritability in Patients With Familial Adenomatous Polyposis With DNA-RNA Paired Testing.

PURPOSE: Patients with germline pathogenic variants (PVs) in APC develop tens (attenuated familial adenomatous polyposis [AFAP]) to innumerable (classic FAP) adenomatous polyps in their colon and are at significantly increased lifetime risk of colorectal cancer. Up to 10% of FAP and up to 50% of patients with AFAP who have undergone DNA-only multigene panel testing (MGPT) do not have an identified PV in APC. We seek to demonstrate how the addition of RNA sequencing run concurrently with DNA can improve detection of germline PVs in individuals with a clinical presentation of AFAP/FAP. METHODS: We performed a retrospective query of individuals tested with paired DNA-RNA MGPT from 2021 to 2022 at a single laboratory and included those with a novel APC PV located in intronic regions infrequently covered by MGPT, a personal history of polyposis, and family medical history provided. All clinical data were deidentified in this institutional review board-exempt study. RESULTS: Three novel APC variants were identified in six families and were shown to cause aberrant splicing because of the creation of a deep intronic cryptic splice site that leads to an RNA transcript subject nonsense-mediated decay. Several carriers had previously undergone DNA-only genetic testing and had received a negative result. CONCLUSION: Here, we describe how paired DNA-RNA MGPT can be used to solve missing heritability in FAP families, which can have important implications in family planning and treatment decisions for patients and their families.

Functional analysis and clinical classification of 462 germline BRCA2 missense variants affecting the DNA binding domain.

Variants of uncertain significance (VUSs) in BRCA2 are a common result of hereditary cancer genetic testing. While more than 4,000 unique VUSs, comprised of missense or intronic variants, have been identified in BRCA2, the few missense variants now classified clinically as pathogenic or likely pathogenic are predominantly located in the region encoding the C-terminal DNA binding domain (DBD). We report on functional evaluation of the influence of 462 BRCA2 missense variants affecting the DBD on DNA repair activity of BRCA2 using a homology-directed DNA double-strand break repair assay. Of these, 137 were functionally abnormal, 313 were functionally normal, and 12 demonstrated intermediate function. Comparisons with other functional studies of BRCA2 missense variants yielded strong correlations. Sequence-based in silico prediction models had high sensitivity, but limited specificity, relative to the homology-directed repair assay. Combining the functional results with clinical and genetic data in an American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP)-like variant classification framework from a clinical testing laboratory, after excluding known splicing variants and functionally intermediate variants, classified 431 of 442 (97.5%) missense variants (129 as pathogenic/likely pathogenic and 302 as benign/likely benign). Functionally abnormal variants classified as pathogenic by ACMG/AMP rules were associated with a slightly lower risk of breast cancer (odds ratio [OR] 5.15, 95% confidence interval [CI] 3.43-7.83) than BRCA2 DBD protein truncating variants (OR 8.56, 95% CI 6.03-12.36). Overall, functional studies of BRCA2 variants using validated assays substantially improved the variant classification yield from ACMG/AMP models and are expected to improve clinical management of many individuals found to harbor germline BRCA2 missense VUS.

Diagnostic Outcomes of Concurrent DNA and RNA Sequencing in Individuals Undergoing Hereditary Cancer Testing.

Importance: Personalized surveillance, prophylaxis, and cancer treatment options for individuals with hereditary cancer predisposition are informed by results of germline genetic testing. Improvements to genomic technology, such as the availability of RNA sequencing, may increase identification of individuals eligible for personalized interventions by improving the accuracy and yield of germline testing. Objective: To assess the cumulative association of paired DNA and RNA testing with detection of disease-causing germline genetic variants and resolution of variants of uncertain significance (VUS). Design, Setting, and Participants: Paired DNA and RNA sequencing was performed on individuals undergoing germline testing for hereditary cancer indication at a single diagnostic laboratory from March 2019 through April 2020. Demographic characteristics, clinical data, and test results were curated as samples were received, and changes to variant classification were assessed over time. Data analysis was performed from May 2020 to June 2023. Main Outcomes and Measures: Main outcomes were increase in diagnostic yield, decrease in VUS rate, the overall results by variant type, the association of RNA evidence with variant classification, and the corresponding predicted effect on cancer risk management. Results: A total of 43 524 individuals were included (median [range] age at testing, 54 [2-101] years; 37 373 female individuals [85.7%], 6224 male individuals [14.3%], and 2 individuals of unknown sex [<0.1%]), with 43 599 tests. A total of 2197 (5.0%) were Ashkenazi Jewish, 1539 (3.5%) were Asian, 3077 (7.1%) were Black, 2437 (5.6%) were Hispanic, 27 793 (63.7%) were White, and 2049 (4.7%) were other race, and for 4507 individuals (10.3%), race and ethnicity were unknown. Variant classification was impacted in 549 individuals (1.3%). Medically significant upgrades were made in 97 individuals, including 70 individuals who had a variant reclassified from VUS to pathogenic/likely pathogenic (P/LP) and 27 individuals who had a novel deep intronic P/LP variant that would not have been detected using DNA sequencing alone. A total of 93 of 545 P/LP splicing variants (17.1%) were dependent on RNA evidence for classification, and 312 of 439 existing splicing VUS (71.1%) were resolved by RNA evidence. Notably, the increase in positive rate (3.1%) and decrease in VUS rate (-3.9%) was higher in Asian, Black, and Hispanic individuals combined compared to White individuals (1.6%; P = .02; and -2.5%; P < .001). Conclusions and Relevance: Findings of this diagnostic study demonstrate that the ability to perform RNA sequencing concurrently with DNA sequencing represents an important advancement in germline genetic testing by improving detection of novel variants and classification of existing variants. This expands the identification of individuals with hereditary cancer predisposition and increases opportunities for personalization of therapeutics and surveillance.

BRCA1 frameshift variants leading to extended incorrect protein C-termini.

Carriers of BRCA1 germline pathogenic variants are at substantially higher risk of developing breast and ovarian cancer than the general population. Accurate identification of at-risk individuals is crucial for risk stratification and the implementation of targeted preventive and therapeutic interventions. Despite significant progress in variant classification efforts, a sizable portion of reported BRCA1 variants remain as variants of uncertain clinical significance (VUS). Variants leading to premature protein termination and loss of essential functional domains are typically classified as pathogenic. However, the impact of frameshift variants that result in an extended incorrect terminus (EIT) is not clear. Using validated functional assays, we conducted a systematic functional assessment of 17 previously reported BRCA1 EIT variants and concluded that 16 constitute loss of function variants. This suggests that most EITs are likely to be pathogenic. However, one variant, c.5578dup, displayed protein expression level, affinity to known binding partners, and activity in transcription and homologous recombination assays comparable to the wild-type BRCA1 protein. Twenty-three additional carriers of c.5578dup were identified at a US clinical diagnostic lab and assessed using a family history likelihood model providing, in combination with the functional data, a likely benign interpretation. These results, consistent with family history data in the current study and available data from ClinVar, indicate that most, but not all, BRCA1 variants leading to EIT constitute loss of function variants, and underscore the need for comprehensive assessment of individual variants.

Characterization of POT1 tumor predisposition syndrome: Tumor prevalence in a clinically diverse hereditary cancer cohort.

PURPOSE: Germline variants in POT1 have been implicated in predisposition to melanoma, sarcoma, and glioma in limited studies. Here, we determine the prevalence of cancer types in individuals with POT1 pathogenic variants (PVs) undergoing multigene panel testing (MGPT) for a broad variety of cancer indications. METHODS: We performed a retrospective review of data provided on clinical documents from individuals with POT1 PVs identified via MGPT over a 5-year period. Tumor prevalence in POT1 PV heterozygotes was compared with MGPT-negative wild-type (WT) controls using χ2 test. RESULTS: POT1 PVs were identified in 227 individuals. POT1 PV and WT (n = 13,315) cohorts had a similar proportion of reported tumors (69.6% and 69.2%, respectively); however, POT1 PV heterozygotes were more likely to be diagnosed with multiple tumors (18.9% vs 8.7%; P < .001). Compared with POT1 WT, we identified a significant increase in melanoma (odds ratio 7.03; 95% CI 4.7-10.5; P < .001) and sarcoma (odds ratio 6.6; 95% CI 3.1-13.9; P < .001). CONCLUSION: This analysis of the largest POT1 PV cohort to date validates the inclusion of POT1 in hereditary cancer MGPT and has the potential to impact clinical management recommendations, particularly for patients and families at risk for melanoma and sarcoma.

Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C.

Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C-/- cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case-control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18-7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71-30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers. SIGNIFICANCE: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants.

Saturation genome editing-based functional evaluation and clinical classification of BRCA2 single nucleotide variants.

Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of surviving endogenously targeted haploid cells. A total of 7013 SNVs were characterized as functionally abnormal (n=955), intermediate/uncertain, or functionally normal (n=5224) based on 95% agreement with ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risk of breast cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). In addition, 86% of tumors associated with abnormal missense SNVs displayed loss of heterozygosity (LOH), whereas 26% of tumors with normal variants had LOH. The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers and the remaining functional scores can be used directly for clinical classification and interpretation of many additional variants. Summary: Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to several types of cancer. However, variants of uncertain significance (VUS) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants to facilitate current and future clinical management of individuals with these variants. Here we show the results from a saturation genome editing (SGE) and functional analysis of all possible single nucleotide variants (SNVs) from exons 15 to 26 that encode the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants. The assay was based on deep sequence analysis of surviving endogenously targeted human haploid HAP1 cells. The assay was calibrated relative to ClinVar known pathogenic and benign missense standards and 95% prevalence thresholds for functionally abnormal and normal variants were identified. Thresholds were validated based on nonsense and synonymous variants. SNVs encoding functionally abnormal missense variants were associated with increased risks of breast and ovarian cancer. The functional assay results were integrated into a ClinGen/ACMG/AMP-like model for clinical classification of the majority of BRCA2 SNVs as pathogenic/likely pathogenic or benign/likely benign. The classified variants can be used for improved clinical management of variant carriers.

Differences in Cancer Phenotypes Among Frequent CHEK2 Variants and Implications for Clinical Care-Checking CHEK2.

Importance: Germline CHEK2 pathogenic variants (PVs) are frequently detected by multigene cancer panel testing (MGPT), but our understanding of PVs beyond c.1100del has been limited. Objective: To compare cancer phenotypes of frequent CHEK2 PVs individually and collectively by variant type. Design, Setting, and Participants: This retrospective cohort study was carried out in a single diagnostic testing laboratory from 2012 to 2019. Overall, 3783 participants with CHEK2 PVs identified via MGPT were included. Medical histories of cancer in participants with frequent PVs, negative MGPT (wild type), loss-of-function (LOF), and missense were compared. Main Outcomes and Measures: Participants were stratified by CHEK2 PV type. Descriptive statistics were summarized including median (IQR) for continuous variables and proportions for categorical characteristics. Differences in age and proportions were assessed with Wilcoxon rank sum and Fisher exact tests, respectively. Frequencies, odds ratios (ORs), 95% confidence intervals were calculated, and P values were corrected for multiple comparisons where appropriate. Results: Of the 3783 participants with CHEK2 PVs, 3473 (92%) were female and most reported White race. Breast cancer was less frequent in participants with p.I157T (OR, 0.66; 95% CI, 0.56-0.78; P<.001), p.S428F (OR, 0.59; 95% CI. 0.46-0.76; P<.001), and p.T476M (OR, 0.74; 95% CI, 0.56-0.98; P = .04) PVs compared with other PVs and an association with nonbreast cancers was not found. Following the exclusion of p.I157T, p.S428F, and p.T476M, participants with monoallelic CHEK2 PV had a younger age at first cancer diagnosis (P < .001) and were more likely to have breast (OR, 1.83; 95% CI, 1.66-2.02; P < .001), thyroid (OR, 1.63; 95% CI, 1.26-2.08; P < .001), and kidney cancer (OR, 2.57; 95% CI, 1.75-3.68; P < .001) than the wild-type cohort. Participants with a CHEK2 PV were less likely to have a diagnosis of colorectal cancer (OR, 0.62; 95% CI, 0.51-0.76; P < .001) compared with those in the wild-type cohort. There were no significant differences between frequent CHEK2 PVs and c.1100del and no differences between CHEK2 missense and LOF PVs. Conclusions and Relevance: CHEK2 PVs, with few exceptions (p.I157T, p.S428F, and p.T476M), were associated with similar cancer phenotypes irrespective of variant type. CHEK2 PVs were not associated with colorectal cancer, but were associated with breast, kidney, and thyroid cancers. Compared with other CHEK2 PVs, the frequent p.I157T, p.S428F, and p.T476M alleles have an attenuated association with breast cancer and were not associated with nonbreast cancers. These data may inform the genetic counseling and care of individuals with CHEK2 PVs.

Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach.

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.

Universal Germline Panel Testing for Individuals With Pheochromocytoma and Paraganglioma Produces High Diagnostic Yield.

BACKGROUND: Practice guidelines to identify individuals with hereditary pheochromocytomas and paragangliomas (PPGLs) advocate for sequential gene testing strategy guided by specific clinical features and predate the routine use of multigene panel testing (MGPT). OBJECTIVE: To describe results of MGPT for hereditary PPGL in a clinically and ancestrally diverse cohort. SETTING: Commercial laboratory based in the United States. METHODS: Clinical data and test results were retrospectively reviewed in 1727 individuals who had targeted MGPT from August 2013 through December 2019 because of a suspicion of hereditary PPGL. RESULTS: Overall, 27.5% of individuals had a pathogenic or likely pathogenic variant (PV), 9.0% had a variant of uncertain significance, and 63.1% had a negative result. Most PVs were identified in SDHB (40.4%), followed by SDHD (21.1%), SDHA (10.1%), VHL (7.8%), SDHC (6.7%), RET (3.7%), and MAX (3.6%). PVs in FH, MEN1, NF1, SDHAF2, and TMEM127 collectively accounted for 6.5% of PVs. Clinical predictors of a PV included extra-adrenal location, early age of onset, multiple tumors, and positive family history of PPGL. Individuals with extra-adrenal PGL and a positive family history were the most likely to have a PV (85.9%). Restricting genetic testing to SDHB/C/D misses one-third (32.8%) of individuals with PVs. CONCLUSION: Our data demonstrate a high diagnostic yield in individuals with and without established risk factors, a low inconclusive result rate, and a substantial contribution to diagnostic yield from rare genes. These findings support universal testing of all individuals with PPGL and the use of concurrent MGPT as the ideal platform.

Classification of the canonical splice alteration MUTYH c.934-2A > G is likely benign based on RNA and clinical data.

MUTYH-associated polyposis (MAP) is an autosomal recessive disorder characterized by the development of multiple adenomatous colonic polyps and an increased lifetime risk of colorectal cancer. Germline biallelic pathogenic variants in MUTYH are responsible for MAP. The MUTYH c.934-2A > G (NM_001128425.1) variant, which is also known as c.850-2A > G for NM_001048174.2, has been identified in our laboratory in more than 800 patients, including homozygous and compound heterozygote carriers. The variant was initially classified as a variant of uncertain significance (VUS) because of lack of a MAP phenotype in biallelic carriers. In two unrelated female patients who were heterozygous carriers of this variant, further testing by RNA sequencing identified an aberrant transcript with a deletion of 9 nt at the start of exon 11 (MUTYH r.934_942del9). This event is predicted to lead to an in-frame loss of three amino acids in a noncritical domain of the protein. This was the only splice defect identified in these patients that was not present in the controls, and the aberrant transcript is derived exclusively from the variant allele, strongly supporting the cause of this splice defect as being the intronic variant, MUTYH c.934-2A > G. The splicing analysis demonstrating a small in-frame skipping of three amino acids in a noncritical domain, along with the absence of a MAP phenotype in our internal cohort of biallelic carriers, provides evidence that the variant is likely benign and not of clinical significance.

Closing the gap: Systematic integration of multiplexed functional data resolves variants of uncertain significance in BRCA1, TP53, and PTEN.

Clinical interpretation of missense variants is challenging because the majority identified by genetic testing are rare and their functional effects are unknown. Consequently, most variants are of uncertain significance and cannot be used for clinical diagnosis or management. Although not much can be done to ameliorate variant rarity, multiplexed assays of variant effect (MAVEs), where thousands of single-nucleotide variant effects are simultaneously measured experimentally, provide functional evidence that can help resolve variants of unknown significance (VUSs). However, a rigorous assessment of the clinical value of multiplexed functional data for variant interpretation is lacking. Thus, we systematically combined previously published BRCA1, TP53, and PTEN multiplexed functional data with phenotype and family history data for 324 VUSs identified by a single diagnostic testing laboratory. We curated 49,281 variant functional scores from MAVEs for these three genes and integrated four different TP53 multiplexed functional datasets into a single functional prediction for each variant by using machine learning. We then determined the strength of evidence provided by each multiplexed functional dataset and reevaluated 324 VUSs. Multiplexed functional data were effective in driving variant reclassification when combined with clinical data, eliminating 49% of VUSs for BRCA1, 69% for TP53, and 15% for PTEN. Thus, multiplexed functional data, which are being generated for numerous genes, are poised to have a major impact on clinical variant interpretation.

Strong functional data for pathogenicity or neutrality classify BRCA2 DNA-binding-domain variants of uncertain significance.

Determination of the clinical relevance of rare germline variants of uncertain significance (VUSs) in the BRCA2 cancer predisposition gene remains a challenge as a result of limited availability of data for use in classification models. However, laboratory-based functional data derived from validated functional assays of known sensitivity and specificity may influence the interpretation of VUSs. We evaluated 252 missense VUSs from the BRCA2 DNA-binding domain by using a homology-directed DNA repair (HDR) assay and identified 90 as non-functional and 162 as functional. The functional assay results were integrated with other available data sources into an ACMG/AMP rules-based classification framework used by a hereditary cancer testing laboratory. Of the 186 missense variants observed by the testing laboratory, 154 were classified as VUSs without functional data. However, after applying protein functional data, 86% (132/154) of the VUSs were reclassified as either likely pathogenic/pathogenic (39/132) or likely benign/benign (93/132), which impacted testing results for 1,900 individuals. These results indicate that validated functional assay data can have a substantial impact on VUS classification and associated clinical management for many individuals with inherited alterations in BRCA2.

Curated multiple sequence alignment for the Adenomatous Polyposis Coli (APC) gene and accuracy of in silico pathogenicity predictions.

Computational algorithms are often used to assess pathogenicity of Variants of Uncertain Significance (VUS) that are found in disease-associated genes. Most computational methods include analysis of protein multiple sequence alignments (PMSA), assessing interspecies variation. Careful validation of PMSA-based methods has been done for relatively few genes, partially because creation of curated PMSAs is labor-intensive. We assessed how PMSA-based computational tools predict the effects of the missense changes in the APC gene, in which pathogenic variants cause Familial Adenomatous Polyposis. Most Pathogenic or Likely Pathogenic APC variants are protein-truncating changes. However, public databases now contain thousands of variants reported as missense. We created a curated APC PMSA that contained >3 substitutions/site, which is large enough for statistically robust in silico analysis. The creation of the PMSA was not easily automated, requiring significant querying and computational analysis of protein and genome sequences. Of 1924 missense APC variants in the NCBI ClinVar database, 1800 (93.5%) are reported as VUS. All but two missense variants listed as P/LP occur at canonical splice or Exonic Splice Enhancer sites. Pathogenicity predictions by five computational tools (Align-GVGD, SIFT, PolyPhen2, MAPP, REVEL) differed widely in their predictions of Pathogenic/Likely Pathogenic (range 17.5-75.0%) and Benign/Likely Benign (range 25.0-82.5%) for APC missense variants in ClinVar. When applied to 21 missense variants reported in ClinVar and securely classified as Benign, the five methods ranged in accuracy from 76.2-100%. Computational PMSA-based methods can be an excellent classifier for variants of some hereditary cancer genes. However, there may be characteristics of the APC gene and protein that confound the results of in silico algorithms. A systematic study of these features could greatly improve the automation of alignment-based techniques and the use of predictive algorithms in hereditary cancer genes.

Rare BRIP1 Missense Alleles Confer Risk for Ovarian and Breast Cancer.

Germline loss-of-function mutations in BRCA1 interacting protein C-terminal helicase 1 (BRIP1) are associated with ovarian carcinoma and may also contribute to breast cancer risk, particularly among patients who develop disease at an early age. Normal BRIP1 activity is required for DNA interstrand cross-link (ICL) repair and is thus central to the maintenance of genome stability. Although pathogenic mutations have been identified in BRIP1, genetic testing more often reveals missense variants, for which the impact on molecular function and subsequent roles in cancer risk are uncertain. Next-generation sequencing of germline DNA in 2,160 early-onset breast cancer and 1,199 patients with ovarian cancer revealed nearly 2% of patients carry a very rare missense variant (minor allele frequency < 0.0001) in BRIP1. This is 3-fold higher than the frequency of all rare BRIP1 missense alleles reported in more than 60,000 individuals of the general population (P < 0.0001, χ 2 test). Using CRISPR-Cas9 gene editing technology and rescue assays, we functionally characterized 20 of these missense variants, focusing on the altered protein's ability to repair ICL damage. A total of 75% of the characterized variants rendered the protein hypomorph or null. In a clinical cohort of >117,000 patients with breast and ovarian cancer who underwent panel testing, the combined OR associated with BRIP1 hypomorph or null missense carriers compared with the general population was 2.30 (95% confidence interval, 1.60-3.30; P < 0.0001). These findings suggest that novel missense variants within the helicase domain of BRIP1 may confer risk for both breast and ovarian cancer and highlight the importance of functional testing for additional variants. SIGNIFICANCE: Functional characterization of rare variants of uncertain significance in BRIP1 revealed that 75% demonstrate loss-of-function activity, suggesting rare missense alleles in BRIP1 confer risk for both breast and ovarian cancer.

Assessment of Diagnostic Outcomes of RNA Genetic Testing for Hereditary Cancer.

Importance: Performing DNA genetic testing (DGT) for hereditary cancer genes is now a well-accepted clinical practice; however, the interpretation of DNA variation remains a challenge for laboratories and clinicians. Adding RNA genetic testing (RGT) enhances DGT by clarifying the clinical actionability of hereditary cancer gene variants, thus improving clinicians' ability to accurately apply strategies for cancer risk reduction and treatment. Objective: To evaluate whether RGT is associated with improvement in the diagnostic outcome of DGT and in the delivery of personalized cancer risk management for patients with hereditary cancer predisposition. Design, Setting, and Participants: Diagnostic study in which patients and/or families with inconclusive variants detected by DGT in genes associated with hereditary breast and ovarian cancer, Lynch syndrome, and hereditary diffuse gastric cancer sent blood samples for RGT from March 2016 to April 2018. Clinicians who ordered genetic testing and received a reclassification report for these variants were surveyed to assess whether RGT-related variant reclassifications changed clinical management of these patients. To quantify the potential number of tested individuals who could benefit from RGT, a cohort of 307 812 patients who underwent DGT for hereditary cancer were separately queried to identify variants predicted to affect splicing. Data analysis was conducted from March 2016 and September 2018. Main Outcomes and Measures: Variant reclassification outcomes following RGT, clinical management changes associated with RGT-related variant reclassifications, and the proportion of patients who would likely be affected by a concurrent DGT and RGT multigene panel testing approach. Results: In total, 93 if 909 eligible families (10.2%) submitted samples for RGT. Evidence from RGT clarified the interpretation of 49 of 56 inconclusive cases (88%) studied; 26 (47%) were reclassified as clinically actionable and 23 (41%) were clarified as benign. Variant reclassifications based on RGT results changed clinical management recommendations for 8 of 18 patients (44%) and 14 of 18 families (78%), based on responses from 18 of 45 clinicians (40%) surveyed. A total of 7265 of 307 812 patients who underwent DGT had likely pathogenic variants or variants of uncertain significance potentially affecting splicing, indicating that approximately 1 in 43 individuals could benefit from RGT. Conclusions and Relevance: In this diagnostic study, conducting RNA testing resolved a substantial proportion of variants of uncertain significance in a cohort of individuals previously tested for cancer predisposition by DGT. Performing RGT might change the diagnostic outcome of at least 1 in 43 patients if performed in all individuals undergoing genetic evaluation for hereditary cancer.

Correction: DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes.

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DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes.

PURPOSE: Gross duplications are ambiguous in terms of clinical interpretation due to the limitations of the detection methods that cannot infer their context, namely, whether they occur in tandem or are duplicated and inserted elsewhere in the genome. We investigated the proportion of gross duplications occurring in tandem in breast cancer predisposition genes with the intent of informing their classifications. METHODS: The DNA breakpoint assay (DBA) is a custom, paired-end, next-generation sequencing (NGS) method designed to capture and detect deep-intronic DNA breakpoints in gross duplications in BRCA1, BRCA2, ATM, CDH1, PALB2, and CHEK2. RESULTS: DBA allowed us to ascertain breakpoints for 44 unique gross duplications from 147 probands. We determined that the duplications occurred in tandem in 114 (78%) carriers from this cohort, while the remainder have unknown tandem status. Among the tandem gross duplications that were eligible for reclassification, 95% of them were upgraded to pathogenic. CONCLUSION: DBA is a novel, high-throughput, NGS-based method that informs the tandem status, and thereby the classification of, gross duplications. This method revealed that most gross duplications in the investigated genes occurred in tandem and resulted in a pathogenic classification, which helps to secure the necessary treatment options for their carriers.