Hereditary Cancer Genes Research Articles

Co-occurrence of germline pathogenic variants for different hereditary cancer syndromes in patients with Lynch syndrome.

BackgroundLynch syndrome (LS) is a hereditary condition characterized by a high risk of colorectal cancer, endometrial cancer, and other neoplasia associated with germline alterations in DNA mismatch repair genes. The classical genetic diagnostic strategy for LS consists of the Sanger sequencing of genes associated with the suspected syndrome. Next‐generation sequencing (NGS) enables the simultaneous sequencing of a large number of hereditary cancer genes. Here, we aimed to study whether other germline pathogenic variants of hereditary cancer genes are present in patients with LS.MethodsA cohort of 84 probands with a previous genetic diagnosis of LS by Sanger sequencing was reanalyzed using NGS via a commercial panel of 94 hereditary cancer genes by hybrid capture. The American College of Medical Genetics and Genomics criteria were used to classify the clinical significance of the variants. The findings of NGS were confirmed by Sanger sequencing. When possible, genetic analyses of the new findings in the proband's relatives were also performed by Sanger sequencing.ResultsWe identified five families (6%), out of 84, with at least two germline pathogenic variants conferring to high or moderate risk in different dominant cancer‐predisposing genes: [MLH1‐BRCA2‐NBN], [MLH1‐BRCA1], [MSH2‐ATM], [MSH6‐NF1], and [MLH1‐FANCA]. Interestingly, only one out of these five families exhibited a clinical phenotype associated with the new pathogenic variants. The family with three pathogenic variants of the [MLH1‐BRCA2‐NBN] genes showed a high aggregation of tumors associated with LS and breast and ovarian cancer syndrome.ConclusionsOur results showed that the co‐occurrence of more than one pathogenic variant in cancer‐predisposing genes was remarkable among cases of LS. In most cases, no clinicial manifestations were associated with the secondary pathogenic variants. Further studies are needed to confirm these findings and elucidate their clinical impact. Reanalysis of LS families should be considered only in families with mixed clinical phenotypes.

Comprehensive Mutational Analysis of the BRCA1-Associated DNA Helicase and Tumor-Suppressor FANCJ/BACH1/BRIP1.

FANCJ (BRIP1/BACH1) is a hereditary breast and ovarian cancer (HBOC) gene encoding a DNA helicase. Similar to HBOC genes, BRCA1 and BRCA2, FANCJ is critical for processing DNA inter-strand crosslinks (ICL) induced by chemotherapeutics, such as cisplatin. Consequently, cells deficient in FANCJ or its catalytic activity are sensitive to ICL-inducing agents. Unfortunately, the majority of FANCJ clinical mutations remain uncharacterized, limiting therapeutic opportunities to effectively use cisplatin to treat tumors with mutated FANCJ. Here, we sought to perform a comprehensive screen to identify FANCJ loss-of-function (LOF) mutations. We developed a FANCJ lentivirus mutation library representing approximately 450 patient-derived FANCJ nonsense and missense mutations to introduce FANCJ mutants into FANCJ knockout (K/O) HeLa cells. We performed a high-throughput screen to identify FANCJ LOF mutants that, as compared with wild-type FANCJ, fail to robustly restore resistance to ICL-inducing agents, cisplatin or mitomycin C (MMC). On the basis of the failure to confer resistance to either cisplatin or MMC, we identified 26 missense and 25 nonsense LOF mutations. Nonsense mutations elucidated a relationship between location of truncation and ICL sensitivity, as the majority of nonsense mutations before amino acid 860 confer ICL sensitivity. Further validation of a subset of LOF mutations confirmed the ability of the screen to identify FANCJ mutations unable to confer ICL resistance. Finally, mapping the location of LOF mutations to a new homology model provides additional functional information. IMPLICATIONS: We identify 51 FANCJ LOF mutations, providing important classification of FANCJ mutations that will afford additional therapeutic strategies for affected patients.

Abstract PD10-09: Development of a breast cancer risk assessment model for ATM mutation carriers incorporating tyrer-cuzick and a polygenic risk score (PRS)

Abstract Background: Germline pathogenic variants (PVs) in the moderate penetrance ATM gene confer a roughly 2-fold increased risk for breast cancer. Currently, any woman with an ATM PV meets the >20% lifetime risk threshold for consideration of relatively aggressive screening recommendations, which include initiating screening at younger ages and consideration of breast MRI. We and others have previously shown that breast cancer risks for women with inherited PVs in many hereditary breast cancer genes can be adjusted using PRS data, breast cancer family history, and other clinical information. Herein we show the development of a comprehensive breast cancer risk model for ATM PV carriers incorporating a previously described 86-variant PRS along with family history/clinical information captured by Tyrer-Cuzick V7.02. Methods: This IRB-approved study included de-identified clinical records from 353,809 women of European ancestry who were tested clinically for hereditary cancer risk with a multi-gene panel. Model development analyzed ATM PV carriers (N=2,666) and women negative for breast cancer gene PVs (N=351,143) who were tested between September 2013 and November 2019 (July 2019 for non-carriers). Women with the unusually high risk ATM c.7271 allele were excluded from analysis. Risk estimates incorporating ATM, PRS, and Tyrer-Cuzick were calculated using a fixed-stratified method that accounted for correlations between risk factors in a manner equivalent to multivariable co-estimation. Risk stratification was assessed in an independent cohort of ATM carriers (N=272) who were tested after November 2019 and were not included in model development. Results: We detected significant positive correlation of ATM status with breast cancer family history (p=1.6×10−7). Within ATM PV carriers, we observed positive yet non-significant (at alpha <0.05) correlation between PRS and breast cancer family history (p=0.10); joint effects were co-estimated using the fixed-stratified method. After adjusting for multiple testing, we found no evidence of interaction of ATM status with clinical factors, or PRS with clinical factors within ATM carriers. In an independent cohort, 30.1% of ATM carriers were categorized as having low breast cancer risk (<20%), 58.5% as moderate risk (20-50%), and 11.4% as high risk (>50%). Conclusions: In ATM PV carriers, our comprehensive model allowed for differentiation of ATM PV carriers into low, moderate, and high breast cancer risk categories. Precision breast cancer risk estimation may inform individualized clinical screening and prevention strategies. Citation Format: Shannon Gallagher, Elisha Hughes, Eric Rosenthal, Allison W. Kurian, Susan Domchek, Judy Garber, Braden Probst, Brian Morris, Placede Tshiaba, Benjamin Roa, Thomas P. Slavin, Susanne Wagner, Jeffrey N. Weitzel, Alexander Gutin, Jerry S. Lanchbury, Mark E. Robson. Development of a breast cancer risk assessment model for ATM mutation carriers incorporating tyrer-cuzick and a polygenic risk score (PRS) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD10-09.

Abstract PD10-07: Rna genetic testing improves detection of patients with hereditary breast cancer

Abstract Background: DNA genetic testing is commonly used to inform treatment decisions for breast cancer patients. In recent years, RNA genetic testing has shown promise for increasing the detection of disease-causing variants and decreasing inconclusive results. Here we describe the impact of concurrent DNA and RNA genetic testing on identifying breast cancer patients with germline cancer predisposition who may have been missed by DNA-only testing. Methods: We performed a retrospective review of breast cancer patients who received concurrent DNA and RNA hereditary cancer panel testing between March 2019 and February 2020 at Ambry Genetics. Patients underwent DNA and RNA genetic testing of up to 18 hereditary cancer genes at the discretion of the ordering healthcare provider (APC, ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MSH2, MSH6, MUTYH, NF1, PALB2, PMS2 exons 1-10, PTEN, RAD51C, RAD51D, and TP53). Breast cancer patients with positive results, defined as the presence of a pathogenic or likely pathogenic variant in any of these 18 genes, were selected for inclusion in this study (n=946). Results: Concurrent DNA and RNA genetic testing led to the identification of 23 breast cancer patients with positive results who would have otherwise received inconclusive or negative results with DNA-only testing. These cases represented 2.4% of all positive results reported in the 18 genes studied (n=23/946). The majority of RNA-related positive results occurred in either ATM (n=14) or BRCA1/2 (n=7). The remaining two cases involved alterations in NF1 and PMS2. Guidelines for risk-reducing breast surgery (BRCA1/2) and breast imaging surveillance (ATM, BRCA1/2, NF1) were relevant for 30.4% and 95.7% of patients with RNA-dependent positive results, respectively. In addition, treatment options such as PARP inhibitors or clinical trial eligibility were potentially implicated for 91.3% of RNA-dependent positives (BRCA1/2, ATM). In 16 of the 23 cases, variants would have been detected by DNA-only testing but would have remained inconclusive without supporting RNA data. In the remaining 7 cases, abnormal RNA results led to the identification of pathogenic/likely pathogenic intronic variants beyond the analytical range of DNA testing. Thus, these seven patients would have received negative results from DNA-only testing. Four of these cases involved pathogenic/likely pathogenic intronic variants in ATM and three involved pathogenic intronic variants in BRCA1. Conclusions: One in 41 breast cancer patients who test positive on concurrent DNA and RNA genetic testing would have received negative or inconclusive results from DNA-only testing. These findings demonstrate the impact of a comprehensive diagnostic testing approach that includes concurrent DNA and RNA analysis and highlights the important implications for the personalized management of these breast cancer patients including potential missed opportunities for early detection and prevention of additional cancer and familial testing in the absence of RGT. Citation Format: Holly LaDuca, Lily Hoang, Carolyn Horton, Jessica Profato, Tameron Harvell, Jill S Dolinsky, Tina Pesaran, Rachid Karam. Rna genetic testing improves detection of patients with hereditary breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD10-07.

Abstract PD10-03: Triple-negative breast cancer (TNBC) risk with pathogenic variants (PV) in hereditary cancer predisposition genes

Abstract BACKGROUND: TNBC is among the most aggressive subtypes of invasive breast cancer (BC), and accounts for approximately 10-15% of incidental BC diagnoses. TNBC is associated with early age of onset (median age of diagnosis <50) and disproportionately affects African American women. Breast MRI is currently recommended to screen for BC in women with at least a moderate-to-high lifetime risk of BC (a 2-fold or higher increased risk), and may also be superior to mammogram to screen for TNBC. TNBC has been most closely associated with germline PVs in BRCA1. However, recent studies have suggested that PVs in other genes previously associated with invasive BC may specifically confer high risks of the TNBC subtype. METHODS: Results were analyzed from 627,219 women undergoing clinical multi-gene panel testing at a single US-based commercial laboratory between 5/2013 and 2/2020, including genes associated with hereditary BC and other cancers. Demographic and personal/family history data were collected on a test requisition form. Individuals who had single- or founder-site testing, or prior BRCA1 or BRCA2 testing, were excluded. Multivariable regression analysis was used to examine the association between PVs/suspected PVs and personal history (PHx) of TNBC. Models were adjusted for age, personal/family cancer history, and ancestry. Odds ratios (OR) with 95% confidence intervals (CI) excluding 1.0 were considered significant. RESULTS: In total, 22.4% (140,467/627,219) of women tested reported PHx of BC, of whom 12.8% (17,951/140,467) reported PHx of TNBC. Elevated risks of TNBC were identified in carriers of PVs in 10 genes (see Table). While the highest TNBC risk was associated with PVs in BRCA1 (OR 21.24, 95% CI 19.71-22.88), high risks were also seen for BARD1 (OR 7.05, 95% CI 5.71-8.71), TP53 (OR 5.64, 95% CI 3.08-10.33), PTEN (OR 5.52, 95% CI 2.35-13.00) and PALB2 (OR 5.27, 95% CI 4.55-6.10). Moderate-to-high risks (2-5-fold increased risk) of TNBC were also seen for carriers of PVs in RAD51C, RAD51D, BRCA2, and CDKN2A/P16. By contrast, PVs in NBN, ATM, and CHEK2 were all associated with an apparent decreased risk of TNBC. CONCLUSIONS: PVs in several hereditary cancer genes routinely tested on multi-gene panel tests are associated with high risks (OR>5.0) and moderate-to-high risks (OR 2.0-5.0) of TNBC. These findings can inform practice guidelines about which genes to test when evaluating breast cancer risk and which PV carriers may benefit from intensive breast screening with magnetic resonance imaging (MRI). Odds ratios for TNBC in germline carriers of PV in hereditary cancer risk genesRisk GenePV Positive with TNBCOR95% CIp-valueHigh RiskBRCA1119321.2419.71-22.88<0.001BARD11257.055.71-8.71<0.001TP53125.643.08-10.33<0.001PTEN65.522.35-13.00<0.001PALB22315.274.55-6.10<0.001Moderate-to-High RiskRAD51C864.923.86-6.26<0.001RAD51D454.643.34-6.45<0.001BRCA24884.434.02-4.89<0.001CDKN2A (p16)172.521.52-4.18<0.001Moderate-to-Low RiskBRIP1771.921.51-2.44<0.001Protective EffectNBN140.550.32-0.940.030ATM400.510.37-0.70<0.001CHEK2490.440.33-0.58<0.001HOXB1350.330.14-0.810.015 Citation Format: Michael J Hall, Eric Rosenthal, Susana San Roman, Ryan Bernhisel, John Kidd, Elisha Hughes, Thomas Slavin, Allison Kurian. Triple-negative breast cancer (TNBC) risk with pathogenic variants (PV) in hereditary cancer predisposition genes [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD10-03.

The Impact of Variants at Branchpoint Splicing Elements in Cancer Genes

Purpose: Branchpoint elements are required for intron removal, and variations at these elements can result in aberrant splicing. We aimed to assess the value of branchpoint annotations generated from recent large-scale studies to select branchpoint-abrogating variants, using hereditary cancer genes as model.Methods: We identified branchpoint elements in 119 genes associated with hereditary cancer from three genome-wide experimentally-inferred and two predicted branchpoint datasets. We then identified variants from public databases that occur within branchpoint elements. We compared conservation, unique variant observations, and population frequencies at different nucleotides within branchpoint motifs. Finally, selected minigene assays were performed to assess the functional impact of variants in branchpoint elements within mismatch repair genes.Results: There was poor overlap between predicted and experimentally-inferred branchpoints. Our analysis of cancer genes suggested that variants at -2nt, -1nt, and branchpoint positions in experimentally-inferred canonical motifs are more likely to be clinically relevant. Minigene assay data showed the -2nt to be more important to branchpoint motif integrity, but also demonstrated fluidity in branchpoint usage.Conclusion: Data from cancer gene analysis suggests that there will be few high-risk alleles that severely impact function via branchpoint abrogation. Results inform a general scheme to prioritize branchpoint motif variants for further study.

Clinical Utility of Functional RNA Analysis for the Reclassification of Splicing Gene Variants in Hereditary Cancer.

Classification of splicing variants (SVs) in genes associated with hereditary cancer is often challenging. The aim of this study was to investigate the occurrence of SVs in hereditary cancer genes and the clinical utility of RNA analysis. 1518 individuals were tested for cancer predisposition, using a Next Generation Sequencing (NGS) panel of 36 genes. Splicing variant analysis was performed using RT-PCR and Sanger Sequencing. In total, 34 different SVs were identified, 53% of which were classified as pathogenic or likely pathogenic. The remaining 16 variants were initially classified as Variant of Uncertain Significance (VUS). RNA analysis was performed for 3 novel variants. The RNA analysis assisted in the reclassification of 20% of splicing variants from VUS to pathogenic. RNA analysis is essential in the case of uncharacterized splicing variants, for proper classification and personalized management of these patients.

Abstract IA-14: Understanding inherited risk for pancreatic cancer

Abstract Pancreatic cancer is the 3rd leading cause of cancer death in the Unites States and the global burden of pancreatic cancer has more than doubled over the past 25 years. Inherited genetic factors explain 22-36% of pancreatic cancer. This includes both high-risk pathogenic variants in hereditary cancer genes and genes associated with hereditary pancreatitis as well as common variants identified in genome-wide association studies. Environmental factors, including cigarette smoking may influence the impact of these genetic variants. High risk pathogenic variants are present in 5-10% of all pancreatic cancer patients and up to 20% of patients with a family history of pancreatic cancer. Studies to identify the genetic basis of the remaining 80% of families with a clustering of pancreatic cancer are ongoing. Identification of the genetic changes that underlie pancreatic cancer not only provide insight into the etiology of this fatal cancer but also provides an opportunity to guide early detection strategies. Citation Format: Alison P. Klein. Understanding inherited risk for pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr IA-14.

317 Poster - Use of multi gene-panel testing to detect hereditary breast cancer gene variants in patients attending to a breast cancer clinic, Peradeniya, Sri Lanka

Background: Globally one in eight women develops breast cancer (BC) in the lifetime. As per the national statistics, 38.7% Sri Lankan women aged 39– 46 years account for BC, which ranked first among all cancers in that age group. About 5–10% of Breast Cancer (BC) cases are clustered in families owing to germline mutations in genes other than BRCA1/2 leading to hereditary breast cancer. Genes in which germline mutations confer highly or moderately increased risks of cancer are called cancer predisposition genes (CPGs). Hereditary breast cancers occur in individuals with germline variants in various CPGs such as BRCA1, BRCA2, CHEK2, ATM, PALB2, TP53, PTEN, STK11, BRIP1, CDKN2A, BARD1, FANC1. Multi-gene panel testing for detection of clinically actionable genetic variants in CPGs are useful in routine clinical practice. This study aimed to describe the pattern of germline variants in a cohort of Sri Lankan patients using multi gene panel testing to assess the cancer risk in individuals. Ethical clearance was obtained from Ethics Review Committee of the Faculty.

Sexual health and menopausal symptoms at one year after risk-reducing salpingo-oophorectomy compared to ovarian retention: The PROSper study

Objective: The purpose of this study is to determine whether risk-reducing salpingo-oophorectomy (RRSO) impairs sexual function and worsens menopausal symptoms compared with ovarian retention among women age 35–50 years with hereditary ovarian cancer gene mutations.

Improving Genetic Testing in Hereditary Cancer by RNA Analysis: Tools to Prioritize Splicing Studies and Challenges in Applying American College of Medical Genetics and Genomics Guidelines

RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants.

Genetic Variant Reinterpretation: Economic and Population Health Management Challenges.

Genetic Variant Reinterpretation: Economic and Population Health Management Challenges.

IDENTIFICATION OF WOMEN AT HIGH RISK FOR GYN HEREDITARY CANCERS MADE SIMPLE

The purpose of this project was to create and trial a screening form to be used in the out-patient setting to identify high-risk carriers for GYN hereditary cancer genes, such as BRCA, Lynch Synd., etc. This screening tool can be applied in internal medicine, OB/GYN and subspecialties, like REI. Early identification of genetic carriers allows for the opportunity to use PGT to prevent transfer of embryos carrying cancer genes when undergoing IVF.

Screening women at high risk for breast cancer: one program fits all? : Subgroup analysis of a large population high risk breast screening program.

The Ontario High Risk Breast Screening program follows women aged 30-69 at an increased risk of breast cancer, using a yearly mammography and breast MRI. The aim of this study is to determine the clinical outcomes for the enrolled women. Observational cohort study following 2081 participants in the high-risk screening program 2011-2017. The participants were divided into three subgroup according to their risk criteria: (a) known carriers of pathogenic variants (PV) in hereditary breast cancer genes. (b) Previous chest radiotherapy. (c) Estimated life time risk (ELR) ≥ 25%, calculated using the International Breast Cancer Intervention Study (IBIS) tool, with no known mutation or previous radiation. All Breast Cancer (BC) diagnosed during the follow-up time were recorded. 673 women carried PVs in hereditary breast cancer genes, 159 had a history of chest radiotherapy, and 1249 had an ELR ≥ 25%. The total cohort of screening years was 8126. Median age at BC diagnosis was 41 for the first group, 47 for the second group and 51 for the third. BC incidence rate was 18.2 for PV mutation carriers, 17.9 for the chest radiotherapy group and 6.2 for ELR ≥ 25%. Hazard ratio was similar for the first two groups, but significantly lower for the ELR ≥ 25% group. When stratifying by age, the incidence rate in the ELR ≥ 25% increased over time, until it became similar to that of the other subgroups after age 50. Our findings question the need to screen women with an elevated lifetime risk using the same screening practices used for women who are PV mutation carriers, or with a history of chest radiation, prior to the age of 50.

Case report expanding the germline AXIN2- related phenotype to include olfactory neuroblastoma and gastric adenoma

BackgroundPathogenic AXIN2 variants cause absence of permanent teeth (hypodontia), sparse hair and eye brows (ectodermal dysplasia), and gastrointestinal polyps and cancer. Inheritance is autosomal dominant with variable penetrance. Only twenty- five patients have been reported from five families. A Mayo Clinic pilot program tested 3009 newly diagnosed cancer patients for pathogenic germline variants in 83 hereditary cancer genes, including AXIN2. We found only one patient with a pathogenic AXIN2 variant.Case presentationThe proband was a 49 year-old female who came to Otolaryngology clinic complaining of right-sided nasal obstruction. Biopsy of identified nasal polyp revealed olfactory neuroblastoma (esthesioneuroblastoma). Surgical resection with gross, total tumor resection was followed by radiation therapy. The patient enrolled in a clinical pilot of genetic testing and a pathogenic variant in AXIN2, c.1822del (p.Leu608Phefs*81) (NM_004655.3) was found. She was seen in Medical Genetics clinic and found to have a personal history of hypodontia. Her eyebrows, hair, and nails were all normal. She underwent upper endoscopy and colonoscopy. A four mm gastric adenoma was found and removed.ConclusionsThis is the first case reported on a patient with a pathogenic, germline AXIN2 variant and an olfactory neuroblastoma or a gastric adenoma. We propose that these could be features of the AXIN2 phenotype. The known association between gastric adenomas and familial adenomatous polyposis, the other Wnt/beta-catenin disorder, supports the hypothesis that pathogenic AXIN2 variants increase risk as well. As the odds of a chance co-occurrence of a pathogenic AXIN2 variant and an olfactory neuroblastoma are so rare, it is worth exploring potential causation. We are building a clinical registry to expand understanding of the AXIN2 phenotype and request any clinicians caring for patients with pathogenic AXIN2 variants to contact us.

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.

Candidate genes for hereditary colorectal cancer: Mutational screening and systematic review.

Genome-wide approaches applied for the identification of new hereditary colorectal cancer (CRC) genes, identified several potential causal genes, including RPS20, IL12RB1, LIMK2, POLE2, MRE11, POT1, FAN1, WIF1, HNRNPA0, SEMA4A, FOCAD, PTPN12, LRP6, POLQ, BLM, MCM9,and the epigenetic inactivation of PTPRJ. Here we attempted to validate the association between variants in these genes and nonpolyposis CRC by performing a mutational screening of the genes and PTPRJ promoter methylation analysis in 473 familial/early-onset CRC cases, a systematic review of the published cases, and assessment of allele frequencies in control population. In the studied cohort, 24 (5%) carriers of (predicted) deleterious variants in the studied genes and no constitutional PTPRJ epimutations were identified. Assessment of allele frequencies in controls compared withfamilial/early-onset patientswith CRC showed association with increased nonpolyposis CRC risk of disruptive variants in RPS20, IL12RB1, POLE2, MRE11 and POT1, and of FAN1 c.149T>G (p.Met50Arg). Lack of association was demonstrated for LIMK2, PTPN12, LRP6, PTPRJ, POLQ, BLM, MCM9 and FOCAD variants. Additional studies are required to provide conclusive evidence for SEMA4A, WIF1, HNRNPA0c.-110G>C, and FOCAD large deletions.

Variant effect on splicing regulatory elements, branchpoint usage, and pseudoexonization: Strategies to enhance bioinformatic prediction using hereditary cancer genes as exemplars.

It is possible to estimate the prior probability of pathogenicity for germline disease gene variants based on bioinformatic prediction of variant effect/s. However, routinely used approaches have likely led to the underestimation and underreporting of variants located outside donor and acceptor splice site motifs that affect messenger RNA (mRNA) processing. This review presents information about hereditary cancer gene germline variants, outside native splice sites, with experimentally validated splicing effects. We list 95 exonic variants that impact splicing regulatory elements (SREs) in BRCA1, BRCA2, MLH1, MSH2, MSH6, and PMS2. We utilized a pre-existing large-scale BRCA1 functional data set to map functional SREs, and assess the relative performance of different tools to predict effects of 283 variants on such elements. We also describe rare examples of intronic variants that impact branchpoint (BP) sites and create pseudoexons. We discuss the challenges in predicting variant effect on BP site usage and pseudoexonization, and suggest strategies to improve the bioinformatic prioritization of such variants for experimental validation. Importantly, our review and analysis highlights the value of considering impact of variants outside donor and acceptor motifs on mRNA splicing and disease causation.

Hereditary Predisposition to Prostate Cancer: From Genetics to Clinical Implications.

Prostate cancer (PrCa) ranks among the top five cancers for both incidence and mortality worldwide. A significant proportion of PrCa susceptibility has been attributed to inherited predisposition, with 10–20% of cases expected to occur in a hereditary/familial context. Advances in DNA sequencing technologies have uncovered several moderate- to high-penetrance PrCa susceptibility genes, most of which have previously been related to known hereditary cancer syndromes, namely the hereditary breast and ovarian cancer (BRCA1, BRCA2, ATM, CHEK2, and PALB2) and Lynch syndrome (MLH1, MSH2, MSH6, and PMS2) genes. Additional candidate genes have also been suggested, but further evidence is needed to include them in routine genetic testing. Recommendations based on clinical features, family history, and ethnicity have been established for more cost-efficient genetic testing of patients and families who may be at an increased risk of developing PrCa. The identification of alterations in PrCa predisposing genes may help to inform screening strategies, as well as treatment options, in the metastatic setting. This review provides an overview of the genetic basis underlying hereditary predisposition to PrCa, the current genetic screening recommendations, and the implications for clinical management of the disease.

Mutation prevalence tables for hereditary cancer derived from multigene panel testing.

Multigene panel testing for cancer predisposition mutations is becoming routine in clinical care. However, the gene content of panels offered by testing laboratories vary significantly, and data on mutation detection rates by gene and by the panel is limited, causing confusion among clinicians on which test to order. Using results from 147,994 multigene panel tests conducted at Ambry Genetics, we built an interactive prevalence tool to explore how differences in ethnicity, age of onset, and personal and family history of different cancers affect the prevalence of pathogenic mutations in 31 cancer predisposition genes, across various clinically available hereditary cancer gene panels. Over 13,000 mutation carriers were identified in this high‐risk population. Most were non‐Hispanic white (74%, n = 109,537), but also Black (n = 10,875), Ashkenazi Jewish (n = 10,464), Hispanic (n = 10,028), and Asian (n = 7,090). The most prevalent cancer types were breast (50%), ovarian (6.6%), and colorectal (4.7%), which is expected based on genetic testing guidelines and clinician referral for testing. The Hereditary Cancer Multi‐Gene Panel Prevalence Tool presented here can be used to provide insight into the prevalence of mutations on a per‐gene and per‐multigene panel basis, while conditioning on multiple custom phenotypic variables to include race and cancer type.