Panel-based next-generation sequencing with copy number variant detection pipeline could increase the detection rate of hereditary colorectal cancer syndrome compared with Sanger sequencing
Aug 2026· Hereditary Cancer in Clinical Practice· 0 citations
TL;DR
Panel-based NGS with CNV analysis was associated with a higher detection rate of clinically relevant variants than phenotype-driven Sanger sequencing in this single-institution cohort, and support the clinical utility of comprehensive germline testing for patients suspected of having hereditary colorectal cancer syndromes.
Abstract
Hereditary colorectal cancer syndromes, such as Lynch syndrome and familial adenomatous polyposis, arise from pathogenic/likely pathogenic (P/LP) germline variants in DNA mismatch repair or tumor suppressor genes. Traditional Sanger sequencing could cover only phenotype-driven genes and cannot detect copy number variants (CNVs). Next-generation sequencing (NGS) enables simultaneous multi-gene analysis and CNV detection. In this study, we compared the results of phenotype-driven Sanger sequencing with those of panel-based NGS in patients suspected of having hereditary colorectal cancer syndromes at a single institution.
Patients tested for hereditary colorectal cancer syndromes between 2008 and 2018 (Sanger) and 2019–2022 (NGS) were retrospectively analyzed. The NGS assay targeted 171 cancer predisposition genes, and CNVs were inferred by read-depth analysis and confirmed with multiplex ligation-dependent probe amplification. Detected variants were classified per ACMG/AMP 2015 guidelines. Microsatellite instability (MSI) and mismatch repair (MMR) immunohistochemistry (IHC) results were compared with molecular findings.
Among 423 patients (254 Sanger, 169 NGS), the detection rate of pathogenic or likely pathogenic variants was higher in NGS (55.0%) than Sanger sequencing (40.6%). NGS additionally identified 17 CNVs (18.1%) that were undetectable by Sanger sequencing and revealed incidental P/LP variants in other cancer-related genes. MSI and MMR IHC results showed strong concordance with molecular findings.
Panel-based NGS with CNV analysis was associated with a higher detection rate of clinically relevant variants than phenotype-driven Sanger sequencing in this single-institution cohort. These findings support the clinical utility of comprehensive germline testing for patients suspected of having hereditary colorectal cancer syndromes, particularly when CNV detection or simultaneous multi-gene analysis is required.
BACKGROUND
Hereditary cancer syndromes (HCSs) account for approximately 5-10% of all cancers and are frequently associated with pathogenic variants (PVs) in genes such as BRCA1 and BRCA2. Nevertheless, a substantial proportion of individuals with strong familial cancer aggregation remain genetically unexplained after standard multigene panel testing. Clinical exome sequencing (CES) may overcome this limitation by enabling a broader exploration of cancer susceptibility genes.
METHODS
This retrospective study included 500 high-risk patients who previously tested negative for BRCA1/2 PVs. All samples were analysed using next-generation sequencing technology. Initially, a 60-gene filtered panel recommended by the American Society of Clinical Oncology (ASCO) guidelines was applied, followed by a 102-gene filtered panel derived from CES to identify additional PVs or likely PVs (LPVs) beyond current diagnostic panels. Variant classification was performed according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines, incorporating ClinGen and gene-specific expert curation recommendations when available.
RESULTS
Within the ASCO panel, PV/LPVs were identified in 9% of patients while 35% carried variants of uncertain significance. CES identified additional PVs/LPVs in 5% of patients in genes not currently included in ASCO-recommended surveillance panels, such as RAD50, BLM, WRN, PMS1 and FANCA. These variants may represent candidate susceptibility loci requiring further clinical and functional validation.
CONCLUSIONS
CES may provide additional exploratory genomic information in selected high-risk BRCA1/2-negative patients, particularly when standard panel testing is uninformative. Although many of the additional findings identified are not yet clinically actionable, CES represents a valuable resource for future reinterpretation as gene-disease evidence evolves. Overall, our findings support the use of CES as a second-tier exploratory approach in carefully selected high-risk cases, while emphasising the need for cautious interpretation and continued gene-disease curation.
Anastasia Dell’Elice, Claudia Palmarini, Federico Anaclerio et al.· Journal of Medical Genetics· 0 citations
Background: Prostate cancer (PCa) is a biologically heterogeneous disease, with a subset of cases driven by hereditary germline alterations. Methods: Targeted germline sequencing using the Twist Fixed Panel was performed in nine high-risk PCa patients (median age 47; range 30–61). Variants were classified based on ClinVar, gnomAD, and in silico prediction tools.
Results: Pathogenic and likely pathogenic variants in DNA damage repair (DDR) genes were identified, including BRCA2, BRIP1, PALB2, and BRCA1. A homozygous ATM variant was observed in one patient. Several ultra-rare variants in MLH3 and BARD1 were also detected. Most variants had allele frequencies <10-6. Conclusions: High-risk and early-onset PCa is associated with rare germline DDR alterations. These findings support expanded germline testing in younger patients and suggest potential relevance for precision oncology approaches, although functional validation is required..
Rawaz Rizgar, Hassan, Abdulkarim Y Karim et al.· Galen medical journal· 0 citations
Lynch syndrome, the most common hereditary cancer syndrome, is caused by germline pathogenic variants in DNA mismatch repair (MMR) genes. Identifying complex or structural MMR gene pathogenic variants can be challenging with short-read sequencing resulting in patients with unexplained MMR-deficient tumours. In this study, we report multiple members of a family who developed MSH2-deficient tumours where clinical multi-gene panel testing of the DNA MMR genes using short-read sequencing did not find a germline pathogenic variant. Oxford Nanopore Technologies adaptive sampling (ONT-AS) long-read sequencing, targeting 104 hereditary cancer genes, identified a shared ~ 3.2 kb SINE-VNTR-Alu (SVA) family F retrotransposon insertion within exon 12 of MSH2 in both the proband and her father. Segregation of this MSH2 SVA insertion by targeted PCR confirmed three additional family members as carriers, including a paternal uncle with three colorectal cancers and an MSH2-deficient sebaceous adenoma. Three of the five heterozygous carriers were cancer affected with at least one MSH2-deficient tumour each. This study demonstrates that long-read sequencing can identify structural variants that are missed by current short-read sequencing multi-gene panel testing, improving the diagnosis of Lynch syndrome. These findings support incorporating long-read sequencing into routine diagnostic workflows for patients with suspected Lynch syndrome following a negative germline test.
Jihoon E. Joo, K. Mahmood, M. Clendenning et al.· Familial Cancer· 0 citations
Familial adenomatous polyposis (FAP) is an autosomal dominant colorectal cancer predisposition syndrome most commonly caused by pathogenic variants in the APC gene. Although targeted gene panel sequencing is routinely used, some FAP cases remain genetically unresolved due to limitations in detecting structural and deep intronic variants. We investigated two unrelated kindreds with clinically evident but genetically unexplained FAP using targeted panel sequencing and whole exome sequencing. Pedigree‐based whole genome sequencing (WGS) was performed to detect cryptic variants, with in silico splice prediction and transcript‐level validation using RT‐PCR, Sanger sequencing, and RT‐qPCR of blood‐derived RNA. WGS uncovered two novel pathogenic APC variants missed by prior testing. In Kindred 1, a heterozygous large deletion encompassing exon 10 was detected and confirmed to cause exon skipping, resulting in a frameshift and premature termination codon. In Kindred 2, a deep intronic complex deletion‐insertion variant was identified between exons 11 and 12, predicted to create novel splice sites. Transcript analysis demonstrated aberrant pseudoexon activation with a 2‐base‐pair deletion, leading to a frameshift and premature termination. Both variants segregated with disease within the respective families and were classified as pathogenic according to ACMG guidelines based on loss‐of‐function effects and functional RNA evidence. Our findings demonstrate that cryptic pathogenic APC variants beyond the detection limits of routine genetic testing can be resolved through WGS combined with transcript‐level validation. This study highlights the importance of transcript‐aware variant interpretation and supports the integration of genome‐wide sequencing and RNA‐based analyses into the diagnostic evaluation of genetically unexplained FAP.
Sung-Ryung Choi, Min Jung Kim, Ja-Lok Ku et al.· Human Mutation· 0 citations
In humans, about 5–10% of all cancers are caused by germline pathogenic variants (PVs) in cancer-predisposing genes, and their identification enables precision oncology approaches, such as surveillance for early detection, preventive medicine, and targeted therapy. Although cancer is a leading cause of death in cats, PVs have not been investigated for precision oncology. We developed a feline-specific multiplex targeted sequencing method to analyze 813 cats for putative PVs in 27 candidate feline cancer-predisposing genes. A total of 784 variants were identified, 13 of which were classified as putative PVs based on predicted truncating impact of amino acid sequence, clinical interpretation of corresponding variants in human, and in silico prediction on amino acid functions. Among 18 cats with one of the 13 putative PVs, seven (38.9%) had various types of confirmed or suspected tumor. Although PV carriers do not always develop cancer even in humans, putative PV carriers without tumors tended to be younger (1.83–16.58, years old, 9.16 years old on average) than the median age of tumor-bearing putative PV carriers (11.83 years old), suggesting that the proportion of affected cats may increase over time. Moreover, five cats with putative PVs in homologous recombination repair genes (BRCA2, RAD51C, or ATM) and two cats with those in mismatch repair genes (MSH2 and MSH6) may be candidates for targeted therapy with PARP inhibitors and immunotherapy with immune checkpoint inhibitors, respectively. These findings provide the first characterization of putative PVs in feline candidate cancer-predisposing genes, representing an important step toward genomics-informed oncology and risk stratification in cats.
A strategy is presented that calculates the median and median absolute deviation of gene-level fold changes across all samples within each sequencing batch and incorporates these measures into the result interpretation, providing batch-level reference metrics and supporting more reliable interpretation in comprehensive genomic profiling.
Chung Lee, Sejoon Lee, Hyun-Hee Koh et al.· Journal of Molecular Diagnos...· 0 citations