Validation of a prostate cancer polygenic risk score.

BACKGROUND: Genome-wide association studies have identified over 100 single-nucleotide polymorphisms (SNPs) associated with prostate cancer (PrCa), and polygenic risk scores (PRS) based on their combined genotypes have been developed for risk stratification. We aimed to assess the contribution of PRS to PrCa risk in a large multisite study. METHODS: The sample included 1972 PrCa cases and 1919 unaffected controls. Next-generation sequencing was used to assess pathogenic variants in 14 PrCa-susceptibility genes and 72 validated PrCa-associated SNPs. We constructed a population-standardized PRS and tested its association with PrCa using logistic regression adjusted for age and family history of PrCa. RESULTS: The mean age of PrCa cases at diagnosis and age of controls at testing/last clinic visit was 59.5 ± 7.2 and 57.2 ± 13.0 years, respectively. Among 1740 cases with pathology data, 57.4% had Gleason score ≤ 6, while 42.6% had Gleason score ≥ 8. In addition, 39.6% cases and 20.1% controls had a family history of PrCa. The PRS was significantly higher in cases than controls (mean ± SD: 1.42 ± 1.11 vs 1.02 ± 0.76; P < .0001). Compared with men in the 1st quartile of age-adjusted PRS, those in the 2nd, 3rd, and 4th quartile were 1.58 (95% confidence interval [CI]: 1.31-1.90), 2.36 (95% CI: 1.96-2.84), and 3.98 (95% CI: 3.29-4.82) times as likely to have PrCa (all P < .0001). Adjustment for family history yielded similar results. PRS predictive performance was consistent with prior literature (area under the receiver operating curve = 0.64; 95% CI: 0.62-0.66). CONCLUSIONS: These data suggest that a 72-SNP PRS is predictive of PrCa, supporting its potential use in clinical risk assessment.

Splicing profile by capture RNA-seq identifies pathogenic germline variants in tumor suppressor genes.

Germline variants in tumor suppressor genes (TSGs) can result in RNA mis-splicing and predisposition to cancer. However, identification of variants that impact splicing remains a challenge, contributing to a substantial proportion of patients with suspected hereditary cancer syndromes remaining without a molecular diagnosis. To address this, we used capture RNA-sequencing (RNA-seq) to generate a splicing profile of 18 TSGs (APC, ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MSH2, MSH6, MUTYH, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, and TP53) in 345 whole-blood samples from healthy donors. We subsequently demonstrated that this approach can detect mis-splicing by comparing splicing profiles from the control dataset to profiles generated from whole blood of individuals previously identified with pathogenic germline splicing variants in these genes. To assess the utility of our TSG splicing profile to prospectively identify pathogenic splicing variants, we performed concurrent capture DNA and RNA-seq in a cohort of 1000 patients with suspected hereditary cancer syndromes. This approach improved the diagnostic yield in this cohort, resulting in a 9.1% relative increase in the detection of pathogenic variants, demonstrating the utility of performing simultaneous DNA and RNA genetic testing in a clinical context.

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.

Comprehensive Paired Tumor/Germline Testing for Lynch Syndrome: Bringing Resolution to the Diagnostic Process.

PURPOSE: The current diagnostic testing algorithm for Lynch syndrome (LS) is complex and often involves multiple follow-up germline and somatic tests. We aimed to describe the results of paired tumor/germline testing performed on a large cohort of patients with colorectal cancer (CRC) and endometrial cancer (EC) to better determine the utility of this novel testing methodology. MATERIALS AND METHODS: We retrospectively reviewed a consecutive series of patients with CRC and EC undergoing paired tumor/germline analysis of the LS genes at a clinical diagnostic laboratory (N = 702). Microsatellite instability, MLH1 promoter hypermethylation, and germline testing of additional genes were performed if ordered. Patients were assigned to one of five groups on the basis of prior tumor screening and germline testing outcomes. Results for each group are described. RESULTS: Overall results were informative regarding an LS diagnosis for 76.1% and 60.8% of patients with mismatch-repair-deficient (MMRd) CRC and EC without and with prior germline testing, respectively. LS germline mutations were identified in 24.8% of patients in the group without prior germline testing, and interestingly, in 9.5% of patients with previous germline testing; four of these were discordant with prior tumor screening. Upon excluding patients with MLH1 promoter hypermethylation and germline mutations, biallelic somatic inactivation was seen in approximately 50% of patients with MMRd tumors across groups. CONCLUSION: Paired testing identified a cause for MMRd tumors in 76% and 61% of patients without and with prior LS germline testing, respectively. Findings support inclusion of tumor sequencing as well as comprehensive LS germline testing in the LS testing algorithm. Paired testing offers a complete, convenient evaluation for LS with high diagnostic resolution.

Association of Breast and Ovarian Cancers With Predisposition Genes Identified by Large-Scale Sequencing.

Importance: Since the discovery of BRCA1 and BRCA2, multiple high- and moderate-penetrance genes have been reported as risk factors for hereditary breast cancer, ovarian cancer, or both; however, it is unclear whether these findings represent the complete genetic landscape of these cancers. Systematic investigation of the genetic contributions to breast and ovarian cancers is needed to confirm these findings and explore potentially new associations. Objective: To confirm reported and identify additional predisposition genes for breast or ovarian cancer. Design, Setting, and Participants: In this sample of 11 416 patients with clinical features of breast cancer, ovarian cancer, or both who were referred for genetic testing from 1200 hospitals and clinics across the United States and of 3988 controls who were referred for genetic testing for noncancer conditions between 2014 and 2015, whole-exome sequencing was conducted and gene-phenotype associations were examined. Case-control analyses using the Genome Aggregation Database as a set of reference controls were also conducted. Main Outcomes and Measures: Breast cancer risk associated with pathogenic variants among 625 cancer predisposition genes; association of identified predisposition breast or ovarian cancer genes with the breast cancer subtypes invasive ductal, invasive lobular, hormone receptor-positive, hormone receptor-negative, and male, and with early-onset disease. Results: Of 9639 patients with breast cancer, 3960 (41.1%) were early-onset cases (≤45 years at diagnosis) and 123 (1.3%) were male, with men having an older age at diagnosis than women (mean [SD] age, 61.8 [12.8] vs 48.6 [11.4] years). Of 2051 women with ovarian cancer, 445 (21.7%) received a diagnosis at 45 years or younger. Enrichment of pathogenic variants were identified in 4 non-BRCA genes associated with breast cancer risk: ATM (odds ratio [OR], 2.97; 95% CI, 1.67-5.68), CHEK2 (OR, 2.19; 95% CI, 1.40-3.56), PALB2 (OR, 5.53; 95% CI, 2.24-17.65), and MSH6 (OR, 2.59; 95% CI, 1.35-5.44). Increased risk for ovarian cancer was associated with 4 genes: MSH6 (OR, 4.16; 95% CI, 1.95-9.47), RAD51C (OR, not estimable; false-discovery rate-corrected P = .004), TP53 (OR, 18.50; 95% CI, 2.56-808.10), and ATM (OR, 2.85; 95% CI, 1.30-6.32). Neither the MRN complex genes nor CDKN2A was associated with increased breast or ovarian cancer risk. The findings also do not support previously reported breast cancer associations with the ovarian cancer susceptibility genes BRIP1, RAD51C, and RAD51D, or mismatch repair genes MSH2 and PMS2. Conclusions and Relevance: The results of this large-scale exome sequencing of patients and controls shed light on both well-established and controversial non-BRCA predisposition gene associations with breast or ovarian cancer reported to date and may implicate additional breast or ovarian cancer susceptibility gene candidates involved in DNA repair and genomic maintenance.

Triple-Negative Breast Cancer Risk Genes Identified by Multigene Hereditary Cancer Panel Testing.

BACKGROUND: Germline genetic testing with hereditary cancer gene panels can identify women at increased risk of breast cancer. However, those at increased risk of triple-negative (estrogen receptor-negative, progesterone receptor-negative, human epidermal growth factor receptor-negative) breast cancer (TNBC) cannot be identified because predisposition genes for TNBC, other than BRCA1, have not been established. The aim of this study was to define the cancer panel genes associated with increased risk of TNBC. METHODS: Multigene panel testing for 21 genes in 8753 TNBC patients was performed by a clinical testing laboratory, and testing for 17 genes in 2148 patients was conducted by a Triple Negative Breast Cancer Consortium (TNBCC) of research studies. Associations between deleterious mutations in cancer predisposition genes and TNBC were evaluated using results from TNBC patients and reference controls. RESULTS: Germline pathogenic variants in BARD1, BRCA1, BRCA2, PALB2, and RAD51D were associated with high risk (odds ratio > 5.0) of TNBC and greater than 20% lifetime risk for overall breast cancer among Caucasians. Pathogenic variants in BRIP1, RAD51C, and TP53 were associated with moderate risk (odds ratio > 2) of TNBC. Similar trends were observed for the African American population. Pathogenic variants in these TNBC genes were detected in 12.0% (3.7% non-BRCA1/2) of all participants. CONCLUSIONS: Multigene hereditary cancer panel testing can identify women with elevated risk of TNBC due to mutations in BARD1, BRCA1, BRCA2, PALB2, and RAD51D. These women can potentially benefit from improved screening, risk management, and cancer prevention strategies. Patients with mutations may also benefit from specific targeted therapeutic strategies.

Quantitative Analysis of BRCA1 and BRCA2 Germline Splicing Variants Using a Novel RNA-Massively Parallel Sequencing Assay.

Clinical genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming widespread. However, the interpretation of variants of unknown significance (VUS) in HBOC genes, such as the clinically actionable genes BRCA1 and BRCA2, remain a challenge. Among the variants that are frequently classified as VUS are those with unclear effects on splicing. In order to address this issue we developed a high-throughput RNA-massively parallel sequencing assay-CloneSeq-capable to perform quantitative and qualitative analysis of transcripts in cell lines and HBOC patients. This assay is based on cloning of RT-PCR products followed by massive parallel sequencing of the cloned transcripts. To validate this assay we compared it to the RNA splicing assays recommended by members of the ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium. This comparison was performed using well-characterized lymphoblastoid cell lines (LCLs) generated from carriers of the BRCA1 or BRCA2 germline variants that have been previously described to be associated with splicing defects. CloneSeq was able to replicate the ENIGMA results, in addition to providing quantitative characterization of BRCA1 and BRCA2 germline splicing alterations in a high-throughput fashion. Furthermore, CloneSeq was used to analyze blood samples obtained from carriers of BRCA1 or BRCA2 germline sequence variants, including the novel uncharacterized alteration BRCA1 c.5152+5G>T, which was identified in a HBOC family. CloneSeq provided a high-resolution picture of all the transcripts induced by BRCA1 c.5152+5G>T, indicating it results in significant levels of exon skipping. This analysis proved to be important for the classification of BRCA1 c.5152+5G>T as a clinically actionable likely pathogenic variant. Reclassifications such as these are fundamental in order to offer preventive measures, targeted treatment, and pre-symptomatic screening to the correct individuals.

False-positive results released by direct-to-consumer genetic tests highlight the importance of clinical confirmation testing for appropriate patient care.

PURPOSE: There is increasing demand from the public for direct-to-consumer (DTC) genetic tests, and the US Food and Drug Administration limits the type of health-related claims DTC tests can market. Some DTC companies provide raw genotyping data to customers if requested, and these raw data may include variants occurring in genes recommended by the American College of Medical Genetics and Genomics to be reported as incidental/secondary findings. The purpose of this study was to review the outcome of requests for clinical confirmation of DTC results that were received by our laboratory and to analyze variant classification concordance. METHODS: We identified 49 patient samples received for further testing that had previously identified genetic variants reported in DTC raw data. For each case identified, information pertaining to the outcome of clinical confirmation testing as well as classification of the DTC variant was collected and analyzed. RESULTS: Our analyses indicated that 40% of variants in a variety of genes reported in DTC raw data were false positives. In addition, some variants designated with the "increased risk" classification in DTC raw data or by a third-party interpretation service were classified as benign at Ambry Genetics as well as several other clinical laboratories, and are noted to be common variants in publicly available population frequency databases. CONCLUSION: Our results demonstrate the importance of confirming DTC raw data variants in a clinical laboratory that is well versed in both complex variant detection and classification.

Diagnostic Exome Sequencing Identifies a Novel Gene, EMILIN1, Associated with Autosomal-Dominant Hereditary Connective Tissue Disease.

Heritable connective tissue diseases are a highly heterogeneous family of over 200 disorders that affect the extracellular matrix. While the genetic basis of several disorders is established, the etiology has not been discovered for a large portion of patients, likely due to rare yet undiscovered disease genes. By performing trio-exome sequencing of a 55-year-old male proband presenting with multiple symptoms indicative of a connective disorder, we identified a heterozygous missense alteration in exon 1 of the Elastin Microfibril Interfacer 1 (EMILIN1) gene, c.64G>A (p.A22T). The proband presented with ascending and descending aortic aneurysms, bilateral lower leg and foot sensorimotor peripheral neuropathy, arthropathy, and increased skin elasticity. Sanger sequencing confirmed that the EMILIN1 alteration, which maps around the signal peptide cleavage site, segregated with disease in the affected proband, mother, and son. The impaired secretion of EMILIN-1 in cells transfected with the mutant p.A22T coincided with abnormal protein accumulation within the endoplasmic reticulum. In skin biopsy of the proband, we detected less EMILIN-1 with disorganized and abnormal coarse fibrils, aggregated deposits underneath the epidermis basal lamina, and dermal cells apoptosis. These findings collectively suggest that EMILIN1 may represent a new disease gene associated with an autosomal-dominant connective tissue disorder.

Enhanced utility of family-centered diagnostic exome sequencing with inheritance model-based analysis: results from 500 unselected families with undiagnosed genetic conditions.

PURPOSE: Diagnostic exome sequencing was immediately successful in diagnosing patients in whom traditional technologies were uninformative. Herein, we provide the results from the first 500 probands referred to a clinical laboratory for diagnostic exome sequencing. METHODS: Family-based exome sequencing included whole-exome sequencing followed by family inheritance-based model filtering, comprehensive medical review, familial cosegregation analysis, and analysis of novel genes. RESULTS: A positive or likely positive result in a characterized gene was identified in 30% of patients (152/500). A novel gene finding was identified in 7.5% of patients (31/416). The highest diagnostic rates were observed among patients with ataxia, multiple congenital anomalies, and epilepsy (44, 36, and 35%, respectively). Twenty-three percent of positive findings were within genes characterized within the past 2 years. The diagnostic rate was significantly higher among families undergoing a trio (37%) as compared with a singleton (21%) whole-exome testing strategy. CONCLUSION: Overall, we present results from the largest clinical cohort of diagnostic exome sequencing cases to date. These data demonstrate the utility of family-based exome sequencing and analysis to obtain the highest reported detection rate in an unselected clinical cohort, illustrating the utility of diagnostic exome sequencing as a transformative technology for the molecular diagnosis of genetic disease.