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

ABSTRACT: 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.

SUBMITTER: Landrith T

PROVIDER: S-EPMC7039900 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Landrith Tyler T Li Bing B Cass Ashley A AA Conner Blair R BR LaDuca Holly H McKenna Danielle B DB Maxwell Kara N KN Domchek Susan S Morman Nichole A NA Heinlen Christopher C Wham Deborah D Koptiuch Cathryn C Vagher Jennie J Rivera Ragene R Bunnell Ann A Patel Gayle G Geurts Jennifer L JL Depas Morgan M MM Gaonkar Shraddha S Pirzadeh-Miller Sara S Krukenberg Rebekah R Seidel Meredith M Pilarski Robert R Farmer Meagan M Pyrtel Khateriaa K Milliron Kara K Lee John J Hoodfar Elizabeth E Nathan Deepika D Ganzak Amanda C AC Wu Sitao S Vuong Huy H Xu Dong D Arulmoli Aarani A Parra Melissa M Hoang Lily L Molparia Bhuvan B Fennessy Michele M Fox Susanne S Charpentier Sinead S Burdette Julia J Pesaran Tina T Profato Jessica J Smith Brandon B Haynes Ginger G Dalton Emily E Crandall Joy Rae-Radecki JR Baxter Ruth R Lu Hsiao-Mei HM Tippin-Davis Brigette B Elliott Aaron A Chao Elizabeth E Karam Rachid R

NPJ precision oncology 20200224

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, CDH ...[more]

PMID: 32133419

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Project description:ImportanceNeuroblastoma accounts for 12% of childhood cancer deaths. The genetic contribution of rare pathogenic germline variation in patients without a family history remains unclear.ObjectiveTo define the prevalence, spectrum, and clinical significance of pathogenic germline variation in cancer predisposition genes (CPGs) in neuroblastoma patients.Design setting and participantsGermline DNA sequencing was performed on the peripheral blood from 786 neuroblastoma patients unselected for family history. Rare variants mapping to CPGs were evaluated for pathogenicity and the percentage of cases harboring pathogenic (P) or likely pathogenic (LP) variants was quantified. The frequency of CPG P-LP variants in neuroblastoma cases was compared to two distinct cancer-free control cohorts to assess enrichment. Matched tumor DNA sequencing was evaluated for "second hits" at CPGs and germline DNA array data from 5,585 neuroblastoma cases and 23,505 cancer-free control children was analyzed to identify rare germline copy number variants (CNVs) affecting genes with an excess burden of P-LP variants in neuroblastoma. Neuroblastoma patients with germline P-LP variants were compared to those without P-LP variants to test for association with clinical characteristics, tumor features, and patient survival.Main outcomes and measuresRare variant prevalence, pathogenicity, enrichment, and association with clinical characteristics, tumor features, and patient survival.ResultsWe observed 116 P-LP variants in CPGs involving 13.9% (109/786) of patients, representing a significant excess burden of P-LP variants compared to controls (9.1%; P = 5.14 × 10-5, Odds Ratio: 1.60, 95% confidence interval: 1.27-2.00). BARD1 harbored the most significant burden of P-LP variants compared to controls (1.0% vs. 0.03%; P = 8.18 × 10-7; Odds Ratio: 32.30, 95% confidence interval: 6.44-310.35). Rare germline CNVs disrupting BARD1 were also identified in neuroblastoma patients (0.05%) but absent in controls (P = 7.08 × 10-3; Odds Ratio: 29.47, 95% confidence interval: 1.52 - 570.70). Overall, P-LP variants in DNA repair genes in this study were enriched in cases compared to controls (8.1% vs. 5.7%; P = 0.01; Odds Ratio: 1.45, 95% confidence interval: 1.08-1.92). Neuroblastoma patients harboring a germline P-LP variant had a worse overall survival when compared to patients without P-LP variants (P = 8.6 × 10-3), and this remained significant in a multivariate Cox proportional-hazards model (P = 0.01).Conclusions and relevanceNeuroblastoma patients harboring germline P-LP variants in CPGs have worse overall survival and BARD1 is an important predisposition gene affected by both common and rare pathogenic variation. Germline sequencing should be performed for all neuroblastoma patients at diagnosis to inform genetic counseling and support future longitudinal and mechanistic studies. Patients with a germline P-LP variant should be closely monitored, regardless of risk group assignment.

Project description:BackgroundAlternative splicing is a ubiquitous post-transcriptional regulation mechanism in most eukaryotic genes. Aberrant splicing isoforms and abnormal isoform ratios can contribute to cancer development. Kinase genes are key regulators of multiple cellular processes. Many kinases are found to be oncogenic and have been intensively investigated in the study of cancer and drugs. RNA-Seq provides a powerful technology for genome-wide study of alternative splicing in cancer besides the conventional gene expression profiling. But this potential has not been fully demonstrated yet.MethodsWe characterized the transcriptome profile of prostate cancer using RNA-Seq data from viewpoints of both differential expression and differential splicing, with an emphasis on kinase genes and their splicing variations. We built a pipeline to conduct differential expression and differential splicing analysis, followed by functional enrichment analysis. We performed kinase domain analysis to identify the functionally important candidate kinase gene in prostate cancer, and calculated the expression levels of isoforms to explore the function of isoform switching of kinase genes in prostate cancer.ResultsWe identified distinct gene groups from differential expression and splicing analyses, which suggested that alternative splicing adds another level to gene expression regulation. Enriched GO terms of differentially expressed and spliced kinase genes were found to play different roles in regulation of cellular metabolism. Function analysis on differentially spliced kinase genes showed that differentially spliced exons of these genes are significantly enriched in protein kinase domains. Among them, we found that gene CDK5 has isoform switching between prostate cancer and benign tissues, which may affect cancer development by changing androgen receptor (AR) phosphorylation. The observation was validated in another RNA-Seq dataset of prostate cancer cell lines.ConclusionsOur work characterized the expression and splicing profiles of kinase genes in prostate cancer and proposed a hypothetical model on isoform switching of CDK5 and AR phosphorylation in prostate cancer. These findings bring new understanding to the role of alternatively spliced kinases in prostate cancer and also demonstrate the use of RNA-Seq data in studying alternative splicing in cancer.

Dataset Information

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

Publications

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

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