Identification of pathogenic variants in cancer genes using base editing screens with editing efficiency correction

AbstractBackgroundMillions of nucleotide variants are identified through cancer genome sequencing and it is clinically important to identify the pathogenic variants among them. By introducing base substitutions at guide RNA target regions in the genome, CRISPR-Cas9-based base editors provide the possibility for evaluating a large number of variants in their genomic context. However, the variability in editing efficiency and the complexity of outcome mapping are two existing problems for assigning guide RNA effects to variants in base editing screens.ResultsTo improve the identification of pathogenic variants, we develop a framework to combine base editing screens with sgRNA efficiency and outcome mapping. We apply the method to evaluate more than 9000 variants across all the exons ofBRCA1andBRCA2genes. Our efficiency-corrected scoring model identifies 910 loss-of-function variants forBRCA1/2, including 151 variants in the noncoding part of the genes such as the 5′ untranslated regions. Many of them are identified in cancer patients and are reported as “benign/likely benign” or “variants of uncertain significance” by clinicians. Our data suggest a need to re-evaluate their clinical significance, which may be helpful for risk assessment and treatment of breast and ovarian cancer.ConclusionsOur results suggest that base editing screens with efficiency correction is a powerful strategy to identify pathogenic variants in a high-throughput manner. Applying this strategy to assess variants in both coding and noncoding regions of the genome could have a direct impact on the interpretation of cancer variants.

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A scalable EHR-based approach for phenotype discovery and variant interpretation for hereditary cancer genes

10.1101/2021.03.18.21253763 ◽

2021 ◽

Author(s):

Chenjie Zeng ◽

Lisa A Bastarache ◽

Ran Tao ◽

Eric Venner ◽

Scott Hebbring ◽

...

Keyword(s):

Hereditary Cancer ◽

Meta Analysis ◽

Genetic Disorders ◽

Genomic Medicine ◽

Risk Scores ◽

Cancer Genes ◽

Pathogenic Variants ◽

Emerge Network ◽

Uncertain Significance ◽

Potential Pathogenicity

Knowledge of the clinical spectrum of rare genetic disorders helps in disease management and variant pathogenicity interpretation. Leveraging electronic health record (EHR)-linked genetic testing data from the eMERGE network, we determined the associations between a set of 23 hereditary cancer genes and 3017 phenotypes in 23544 individuals. This phenome-wide association study replicated 45% (184/406) of known gene-phenotype associations (P = 5.1 ×10-125). Meta-analysis with an independent EHR-derived cohort of 3242 patients confirmed 14 novel associations with phenotypes in the neoplastic, genitourinary, digestive, congenital, metabolic, mental and neurologic categories. Phenotype risk scores (PheRS) based on weighted aggregations of EHR phenotypes accurately predicted variant pathogenicity for at least 50% of pathogenic variants for 8/23 genes. We generated a catalog of PheRS for 7800 variants, including 5217 variants of uncertain significance, to provide empirical evidence of potential pathogenicity. This study highlights the potential of EHR data in genomic medicine.

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Comparison of Somatic and Germline Variant Interpretation in Hereditary Cancer Genes

JCO Precision Oncology ◽

10.1200/po.19.00144 ◽

2019 ◽

pp. 1-8

Author(s):

Emily W. Moody ◽

Jennie Vagher ◽

Whitney Espinel ◽

David Goldgar ◽

Kelsi J. Hagerty ◽

...

Keyword(s):

Health Care Providers ◽

Hereditary Cancer ◽

Clinical Test ◽

Variant Interpretation ◽

Test Results ◽

Cancer Genes ◽

Care Providers ◽

Pathogenic Variants ◽

Uncertain Significance ◽

Brca1 Brca2

PURPOSE To compare the classification of genetic variants reported on tumor genomic profiling (TGP) reports with germline classifications on clinical test results and ClinVar. Results will help to inform germline testing discussions and decisions in patients with tumor variants in genes that are relevant to hereditary cancer risk. PATIENTS AND METHODS This study compared somatic and germline classifications of small nucleotide variants in the following genes: BRCA1, BRCA2, CHEK2, PALB2, ATM, MLH1, MSH2, MSH6, and PMS2. Somatic classifications were taken from reports from a single commercial TGP laboratory of tests ordered by providers at Huntsman Cancer Institute between March 2014 and June 2018. Somatic variant interpretations were compared with classifications from germline test results as well as with ClinVar interpretations. RESULTS Of the 623 variants identified on TGP, 353 had a definitive classification in ClinVar, and 103 were assayed with a germline test, with 66 of the variants tested observed in germline. Analysis of somatic variants of uncertain significance listed on TGP reports determined that 22% had a different interpretation compared with ClinVar and that 32% differed from the interpretation on a germline test result. Pathogenic variants on TGP test results were found to differ 13% and 5% of the time compared with ClinVar interpretations and germline test results, respectively. CONCLUSION These results suggest that TGP variants are often classified differently in a germline context. Differences may be due to different processes in variant interpretation between somatic and germline laboratories. These results are important for health care providers to consider when making decisions about additional testing for hereditary cancer risks.

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Programmable RNA base editing with a single gRNA-free enzyme

10.1101/2021.08.31.458316 ◽

2021 ◽

Author(s):

Wenjian Han ◽

Wendi Huang ◽

Miaowei Mao ◽

Tong Wei ◽

Yanwen Ye ◽

...

Keyword(s):

Rna Editing ◽

Rna Binding ◽

Artificial Enzyme ◽

Free System ◽

Editing Efficiency ◽

Base Editing ◽

Guide Rna ◽

Human Proteins ◽

Target Effect ◽

Puf Domain

ABSTRACTProgrammable RNA editing enables rewriting gene expression without changing genome sequences. Current tools for specific RNA editing dependent on the assembly of guide RNA into an RNA/protein complex, causing delivery barrier and low editing efficiency. We report a new gRNA-free system, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. This artificial enzyme contains a human-originated programmable PUF domain to specifically recognize RNAs and different deaminase domains to achieve efficient A-to-I or C-to-U editing, which achieved 60-80% editing rate in human cells, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. The RNA-binding domain in REWIREs was further optimized to improve editing efficiency and minimize off-target effects. We applied the REWIREs to correct disease-associated mutations and achieve both types of base editing in mice. As a single-component system originated from human proteins, REWIRE presents a precise and efficient RNA editing platform with broad applicability.

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Predicting RNA splicing from DNA sequence using Pangolin

10.1101/2021.07.06.451243 ◽

2021 ◽

Author(s):

Tony Zeng ◽

Yang I Li

Keyword(s):

Deep Learning ◽

Dna Sequence ◽

Rna Splicing ◽

Rare Variants ◽

Sequence Data ◽

Learning Approaches ◽

Loss Of Function ◽

Pathogenic Variants ◽

Uncertain Significance ◽

The Impact

Recent progress in deep learning approaches have greatly improved the prediction of RNA splicing from DNA sequence. Here, we present Pangolin, a deep learning model to predict splice site strength in multiple tissues that has been trained on RNA splicing and sequence data from four species. Pangolin outperforms state of the art methods for predicting RNA splicing on a variety of prediction tasks. We use Pangolin to study the impact of genetic variants on RNA splicing, including lineage-specific variants and rare variants of uncertain significance. Pangolin predicts loss-of-function mutations with high accuracy and recall, particularly for mutations that are not missense or nonsense (AUPRC = 0.93), demonstrating remarkable potential for identifying pathogenic variants.

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Determining the pathogenicity of variants of uncertain significance and identification of a founder variant in the epilepsy-associated gene, SZT2

10.1101/2021.01.06.425612 ◽

2021 ◽

Author(s):

Jeffrey D Calhoun ◽

Miriam C Aziz ◽

Hannah C Happ ◽

Jonathan Gunti ◽

Colleen Gleason ◽

...

Keyword(s):

Neurodevelopmental Disorder ◽

Scaffolding Protein ◽

Loss Of Function ◽

Variants Of Uncertain Significance ◽

Pathogenic Variants ◽

Large Size ◽

Common Causes ◽

Uncertain Significance ◽

Amino Acid Sensing ◽

Genetic Epilepsies

AbstractBiallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTOR signaling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions. We report a cohort of twelve individuals with biallelic SZT2 variants and phenotypes consistent with SZT2-related neurodevelopmental disorder. The majority of this cohort contained one or more SZT2 variants of uncertain significance (VUS). We developed a novel individualized platform to functionally characterize SZT2 VUSs. We identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis determined this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Overall, we present a FACS-based rapid assay to distinguish pathogenic variants from VUSs in SZT2, using an approach that is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.

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Case series examining somatic test results for patients with hereditary cancer syndromes associated with gastrointestinal cancer risk.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.4_suppl.680 ◽

2020 ◽

Vol 38 (4_suppl) ◽

pp. 680-680

Author(s):

Kristen Pauley ◽

Cathryn Koptiuch ◽

Samantha Greenberg ◽

Gammon Amanda ◽

Christopher Nevala-Plagemann ◽

...

Keyword(s):

Lynch Syndrome ◽

Treatment Options ◽

Case Series ◽

Parp Inhibitors ◽

Cancer Genes ◽

Panel Testing ◽

Pathogenic Variants ◽

Test Criteria ◽

Uncertain Significance ◽

Germline Testing

680 Background: Somatic tumor testing may identify germline pathogenic variants (PV) associated with cancer predisposition syndromes. Labs differ whether they offer somatic only or paired germline analysis. Methods used by somatic testing labs, even those that include germline analysis, differ from designated germline labs that have optimized the identification of germline PV. Methods: Chart reviews were performed for patients who had testing through both somatic and designated germline laboratories. Cases with discrepant results in which germline PV were not detected by the somatic laboratory are summarized. Results: Nine cases with discrepant results. Five had paired germline testing and 4 somatic testing only. All 9 patients met the criteria to undergo designated germline testing, either for Lynch syndrome (3) or BRCA1/2 testing (6), based on personal and/or family history. Designated germline testing identified 4 MLH1, 1 BRCA1, 2 ATM, 1 MUTYH and 1 RAD50 PV not reported by the somatic labs’ tumor or germline analysis; 2 MLH1 PV were called variants of uncertain significance by somatic testing but classified as PV by ClinVar and designated germline labs. Three PV identified by designated germline labs are targets for PARP inhibitors and resulted in different treatment options. Three of the MLH1 PV were identified in patients meeting Lynch Syndrome test criteria while 1 was identified in a patient meeting BRCA1/2 criteria. Among the 5 other patients meeting BRCA1/2 test criteria, 3 had PV in breast cancer genes (2 ATM, 1 BRCA1) and 2 had PV in other cancer genes ( MUTYH and RAD50) not reported by the somatic labs, highlighting the importance of panel testing. Conclusions: Methods used by somatic labs, regardless of inclusion of germline analysis, are not equivalent to those of designated germline labs. Overlooked germline PV may miss identification of hereditary syndromes and targeted therapy opportunities (e.g. Anti-PD1 immunotherapy, PARP inhibitors). Patients meeting criteria for genetic evaluation should be referred for designated germline testing regardless of somatic testing outcomes.

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The Spectrum of DEPDC5-Related Epilepsy

Journal of Pediatric Neurology ◽

10.1055/s-0041-1727139 ◽

2021 ◽

Author(s):

Giulia Salomone ◽

Mattia Comella ◽

Anna Portale ◽

Giulia Pecora ◽

Giuseppe Costanza ◽

...

Keyword(s):

Mammalian Target Of Rapamycin ◽

High Rate ◽

Loss Of Function ◽

Unexpected Death ◽

Neurodevelopmental Outcomes ◽

Pathogenic Variants ◽

Malformation Of Cortical Development ◽

Uncertain Significance ◽

Mtorc1 Pathway ◽

Intrafamilial Variability

AbstractDisheveled EGL-10 and pleckstrin domain-containing protein 5 (DEPDC5) is a key member of the GAP activity toward rags complex 1 complex, which inhibits the mammalian target of rapamycin complex 1 (mTORC1) pathway. DEPDC5 loss-of-function mutations lead to an aberrant activation of the mTOR signaling. At neuronal level, the increased mTOR cascade causes the generation of epileptogenic dysplastic neuronal circuits and it is often associated with malformation of cortical development. The DEPDC5 phenotypic spectrum ranges from sporadic early-onset epilepsies with poor neurodevelopmental outcomes to familial focal epilepsies and sudden unexpected death in epilepsy; a high rate of inter- and intrafamilial variability has been reported. To date, clear genotype–phenotype correlations have not been proven. More studies are required to elucidate the significance of likely pathogenic/variants of uncertain significance. The pursuit of a molecular targeted antiepileptic therapy is a future challenge.

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Germline CDKN1B Loss-of-Function Variants Cause Pediatric Cushing’s Disease With or Without an MEN4 Phenotype

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgaa160 ◽

2020 ◽

Vol 105 (6) ◽

pp. 1983-2005

Author(s):

Fanny Chasseloup ◽

Nathan Pankratz ◽

John Lane ◽

Fabio R Faucz ◽

Margaret F Keil ◽

...

Keyword(s):

Cushing’S Disease ◽

Colon Adenocarcinoma ◽

Copy Number Variant ◽

Cushing's Disease ◽

In Vitro Assays ◽

Loss Of Function ◽

Pathogenic Variants ◽

Uncertain Significance ◽

Cdkn1b Gene

Abstract Context Germline loss-of-function CDKN1B gene variants cause the autosomal dominant syndrome of multiple endocrine neoplasia type 4 (MEN4). Even though pituitary neuroendocrine tumors are a well-known component of the syndrome, only 2 cases of Cushing’s disease (CD) have so far been described in this setting. Aim To screen a large cohort of CD patients for CDKN1B gene defects and to determine their functional effects. Patients We screened 211 CD patients (94.3% pediatric) by germline whole-exome sequencing (WES) only (n = 157), germline and tumor WES (n = 27), Sanger sequencing (n = 6), and/or germline copy number variant (CNV) analysis (n = 194). Sixty cases were previously unpublished. Variant segregation was investigated in the patients’ families, and putative pathogenic variants were functionally characterized. Results Five variants of interest were found in 1 patient each: 1 truncating (p.Q107Rfs*12) and 4 nontruncating variants, including 3 missense changes affecting the CDKN1B protein scatter domain (p.I119T, p.E126Q, and p.D136G) and one 5’ untranslated region (UTR) deletion (c.-29_-26delAGAG). No CNVs were found. All cases presented early (10.5 ± 1.3 years) and apparently sporadically. Aside from colon adenocarcinoma in 1 carrier, no additional neoplasms were detected in the probands or their families. In vitro assays demonstrated protein instability and disruption of the scatter domain of CDKN1B for all variants tested. Conclusions Five patients with CD and germline CDKN1B variants of uncertain significance (n = 2) or pathogenic/likely pathogenic (n = 3) were identified, accounting for 2.6% of the patients screened. Our finding that germline CDKN1B loss-of-function may present as apparently sporadic, isolated pediatric CD has important implications for clinical screening and genetic counselling.

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Case Report: Functional Investigation of an Undescribed Missense Variant Affecting Splicing in a Patient With Dravet Syndrome

Frontiers in Neurology ◽

10.3389/fneur.2021.761892 ◽

2021 ◽

Vol 12 ◽

Author(s):

Peter Sparber ◽

Svetlana Mikhaylova ◽

Varvara Galkina ◽

Yulia Itkis ◽

Mikhail Skoblov

Keyword(s):

Missense Variant ◽

Febrile Seizures ◽

Dravet Syndrome ◽

Loss Of Function ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Pathogenic Variants ◽

Uncertain Significance ◽

Functional Investigation ◽

Coding Variants

Pathogenic variants in the SCN1A gene are associated with a spectrum of epileptic disorders ranging in severity from familial febrile seizures to Dravet syndrome. Large proportions of reported pathogenic variants in SCN1A are annotated as missense variants and are often classified as variants of uncertain significance when no functional data are available. Although loss-of-function variants are associated with a more severe phenotype in SCN1A, the molecular mechanism of single nucleotide variants is often not clear, and genotype-phenotype correlations in SCN1A-related epilepsy remain uncertain. Coding variants can affect splicing by creating novel cryptic splicing sites in exons or by disrupting exonic cis-regulation elements crucial for proper pre-mRNA splicing. Here, we report a novel case of Dravet syndrome caused by an undescribed missense variant, c.4852G>A (p.(Gly1618Ser)). By midigene splicing assay, we demonstrated that the identified variant is in fact splice-affecting. To our knowledge, this is the first report on the functional investigation of a missense variant affecting splicing in Dravet syndrome.

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Investigation of monogenic causes of familial breast cancer: data from the BEACCON case-control study

npj Breast Cancer ◽

10.1038/s41523-021-00279-9 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Na Li ◽

Belle W. X. Lim ◽

Ella R. Thompson ◽

Simone McInerny ◽

Magnus Zethoven ◽

...

Keyword(s):

Breast Cancer ◽

High Risk ◽

Candidate Genes ◽

Familial Aggregation ◽

Loss Of Function ◽

Cancer Data ◽

Pathogenic Variants ◽

New Genes ◽

Predisposing Genes ◽

Predisposition Genes

AbstractBreast cancer (BC) has a significant heritable component but the genetic contribution remains unresolved in the majority of high-risk BC families. This study aims to investigate the monogenic causes underlying the familial aggregation of BC beyond BRCA1 and BRCA2, including the identification of new predisposing genes. A total of 11,511 non-BRCA familial BC cases and population-matched cancer-free female controls in the BEACCON study were investigated in two sequencing phases: 1303 candidate genes in up to 3892 cases and controls, followed by validation of 145 shortlisted genes in an additional 7619 subjects. The coding regions and exon–intron boundaries of all candidate genes and 14 previously proposed BC genes were sequenced using custom designed sequencing panels. Pedigree and pathology data were analysed to identify genotype-specific associations. The contribution of ATM, PALB2 and CHEK2 to BC predisposition was confirmed, but not RAD50 and NBN. An overall excess of loss-of-function (LoF) (OR 1.27, p = 9.05 × 10−9) and missense (OR 1.27, p = 3.96 × 10−73) variants was observed in the cases for the 145 candidate genes. Leading candidates harbored LoF variants with observed ORs of 2–4 and individually accounted for no more than 0.79% of the cases. New genes proposed by this study include NTHL1, WRN, PARP2, CTH and CDK9. The new candidate BC predisposition genes identified in BEACCON indicate that much of the remaining genetic causes of high-risk BC families are due to genes in which pathogenic variants are both very rare and convey only low to moderate risk.

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