Interpretation of BRCA2 Splicing Variants: A Case Series of Challenging Variant Interpretations and the Importance of Functional RNA Analysis

AbstractA substantial proportion of pathogenic variants associated with an increased risk of hereditary cancer are sequence variants affecting RNA splicing. The classification of these variants can be complex when both non-functional and functional transcripts are produced from the variant allele. We present four BRCA2 splice site variants with complex variant interpretations (BRCA2 c.68-3T>G, c.68-2A>G, c.425G>T, c.8331+2T>C). Evidence supporting a pathogenic classification is available for each variant, including in silico models, absence in population databases, and published functional data. However, comprehensive RNA analysis showed that some functional transcript may be produced by each variant. BRCA2 c.68-3T>G results in a partial splice defect. For BRCA2 c.68-2A>G and c.425G>T, aberrant splicing was shown to produce a potentially functional, in-frame transcript. BRCA2 c.8331+2T>C may utilize a functional GC donor in place of the wild-type GT donor. The severity of cancer history for carriers of these variants was also assessed using a history weighting algorithm and was not consistent with pathogenic controls (carriers of known pathogenic variants in BRCA2). Due to the conflicting evidence, our laboratory classifies these BRCA2 variants as variants of uncertain significance. This highlights the importance of evaluating new and existing evidence to ensure accurate variant classification and appropriate patient care.

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Transcriptome analysis provides critical answers to the “variants of uncertain significance” conundrum

10.22541/au.164191366.62767430/v1 ◽

2022 ◽

Author(s):

Mackenzie Postel ◽

Julie O. Culver ◽

Charité Ricker ◽

David Craig

Keyword(s):

Transcriptome Analysis ◽

Critical Role ◽

European Ancestry ◽

Biologically Relevant ◽

Rna Analysis ◽

Pathogenic Variants ◽

Level Data ◽

Splicing Variants ◽

Uncertain Significance ◽

Generation Sequencing

The vast volume of data that has been generated as a result of the next-generation sequencing revolution is overwhelming to sift through and interpret. Parsing functional vs. non-functional and benign vs. pathogenic variants continues to be a challenge. Out of three billion bases, the genomes of two given individuals will only differ by about 3 million variants (0.1%). Furthermore, only a small fraction of these are biologically-relevant and, of those that are functional, only a handful actually drive disease pathology. While whole genome and exome sequencing have transformed our collective understanding of the role that genetics plays in disease pathogenesis, there are certain conditions and populations for whom DNA-level data has failed to produce a molecular diagnosis. Patients of non-White race/non-European ancestry are disproportionately affected by “variants of unknown/uncertain significance” (VUS). This limits the scope of precision medicine for minority patients and perpetuates health disparities. VUS often include deep intronic and splicing variants which are difficult to interpret in DNA alone. RNA analysis is capable of illuminating the consequences of VUS thereby allowing for their reclassification as pathogenic vs. benign. Here we review the critical role, going forward, of transcriptome analysis for clarifying VUS in both neoplastic and non-neoplastic diseases.

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RNA Splicing Defects in Hypertrophic Cardiomyopathy: Implications for Diagnosis and Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms21041329 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1329

Author(s):

Marta Ribeiro ◽

Marta Furtado ◽

Sandra Martins ◽

Teresa Carvalho ◽

Maria Carmo-Fonseca

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Rna Splicing ◽

Clinical Utility ◽

Clinical Diagnostics ◽

Rna Analysis ◽

Uncertain Significance ◽

Associated Proteins ◽

High Throughput Dna Sequencing ◽

Splicing Defects ◽

Diagnostic Outcomes

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is predominantly caused by mutations in genes that encode sarcomere-associated proteins. Effective gene-based diagnosis is critical for the accurate clinical management of patients and their family members. However, the introduction of high-throughput DNA sequencing approaches for clinical diagnostics has vastly expanded the number of variants of uncertain significance, leading to many inconclusive results that limit the clinical utility of genetic testing. More recently, developments in RNA analysis have been improving diagnostic outcomes by identifying new variants that interfere with splicing. This review summarizes recent discoveries of RNA mis-splicing in HCM and provides an overview of research that aims to apply the concept of RNA therapeutics to HCM.

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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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Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance

eLife ◽

10.7554/elife.71137 ◽

2022 ◽

Vol 11 ◽

Author(s):

Hirokazu Kimura ◽

Raymond M Paranal ◽

Neha Nanda ◽

Laura D Wood ◽

James R Eshleman ◽

...

Keyword(s):

Functional Characterization ◽

Ductal Adenocarcinoma ◽

Proliferation Assay ◽

Variants Of Uncertain Significance ◽

Pathogenic Variants ◽

Deleterious Alleles ◽

Increased Risk ◽

Uncertain Significance

Pathogenic germline CDKN2A variants are associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC). CDKN2A variants of uncertain significance (VUSs) are reported in up to 4.3% of patients with PDAC and result in significant uncertainty for patients and their family members as an unknown fraction are functionally deleterious, and therefore, likely pathogenic. Functional characterization of CDKN2A VUSs is needed to reclassify variants and inform clinical management. 29 germline CDKN2A VUSs previously reported in patients with PDAC or in ClinVar were evaluated using a validated in vitro cell proliferation assay. 12 of the 29 CDKN2A VUSs were functionally deleterious (11 VUSs) or potentially functionally deleterious (1 VUS) and were reclassified as likely pathogenic variants. Thus, over 40% of CDKN2A VUSs identified in patients with PDAC are functionally deleterious and likely pathogenic. When incorporating VUSs found to be functionally deleterious, and reclassified as likely pathogenic, the prevalence of pathogenic/likely pathogenic CDKN2A in patients with PDAC reported in the published literature is increased to up to 4.1% of patients, depending on family history. Therefore, CDKN2A VUSs may play a significant, unappreciated role in risk of pancreatic cancer. These findings have significant implications for the counselling and care of patients and their relatives.

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Expanding the phenotype of E318K (c.952G > A) MITF germline mutation carriers: case series and review of the literature

Hereditary Cancer in Clinical Practice ◽

10.1186/s13053-021-00189-8 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Leandro Jonata Carvalho Oliveira ◽

Aline Bobato Lara Gongora ◽

Fabiola Ambrosio Silveira Lima ◽

Felipe Sales Nogueira Amorim Canedo ◽

Carla Vanessa Quirino ◽

...

Keyword(s):

Breast Cancer ◽

Case Series ◽

Analysis Data ◽

Pathogenic Variant ◽

Fallopian Tube Cancer ◽

Case Presentation ◽

Pathogenic Variants ◽

Increased Risk ◽

Total Cancer ◽

Supergene Family

Abstract Background The microphthalmia-associated transcription factor gene (MITF) belongs to the MYC supergene family and plays an important role in melanocytes’ homeostasis. Individuals harboring MITF germline pathogenic variants are at increased risk of developing cancer, most notably melanoma and renal cell carcinoma. Case presentation We describe a cohort of ten individuals who harbor the same MITF c.952G > A (p.Glu 318Lys), or p.E318K, germline pathogenic variant. Six carriers developed at least one malignancy (4 cases of breast cancer; 1 cervical cancer; 1 colon cancer; 1 melanoma; 1 ovarian/fallopian tube cancer). A significant phenotypic heterogeneity was found among these individuals and their relatives. Breast cancer was, overall, the most frequent malignancy observed in this case series, with 13 occurrences of 60 (21.67 %) total cancer cases described among the probands and their relatives. Conclusions Our retrospective analysis data raise the hypothesis of a possible association of the MITF p.E318K pathogenic variant with an increased risk of breast cancer.

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Novel candidates of pathogenic variants of the BRCA1 and BRCA2 genes in a 3,552 Japanese whole-genome sequence dataset (3.5KJPNv2)

10.1101/2020.07.17.208454 ◽

2020 ◽

Author(s):

Hideki Tokunaga ◽

Keita Iida ◽

Atsushi Hozawa ◽

Soichi Ogishima ◽

Yoh Watanabe ◽

...

Keyword(s):

Prospective Cohort ◽

Clinical Evidence ◽

Whole Genome Sequence ◽

Whole Genome ◽

Cancer Syndrome ◽

Brca1 And Brca2 ◽

Cancer History ◽

Pathogenic Variants ◽

Uncertain Significance

AbstractIdentification of pathogenic germline variants yet no clinical evidence in BRCA genes has become important in patient care of hereditary breast and ovarian cancer syndrome (HBOC). Computational scoring and prospective cohort studies may help to identify such pathogenic variants. We annotated the variants in the BRCA1 and BRCA2 genes from a dataset of 3,552 whole-genome sequences obtained from members of the genome cohorts by Tohoku Medical Megabank Project (TMM) with the InterVar software. Computational impact scores (CADD_phred and Eigen_raw) and minor allele frequencies (MAF) of pathogenic (P) and likely pathogenic (LP) variants in ClinVar are used for filtration criteria. Familial predispositions in cancers among the 35,000 TMM genome cohort participants are analyzed to verify the pathogenicity. Seven potentially pathogenic variants were newly identified. Carriers of these potential pathogenic variants and definite P and LP variants among participants of the TMM prospective cohort show a statistically significant preponderance in cancer onset in sisters in the self-reported cancer history. Filtering by computational scoring and MAF is useful to identify potential pathogenic variants in BRCA genes for Japanese population. These results will be helpful to follow up the carriers of variants of uncertain significance in the HBOC genes.

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Prevalence of Germline Pathogenic Variants in Cancer Predisposing Genes in Czech and Belgian Pancreatic Cancer Patients

Cancers ◽

10.3390/cancers13174430 ◽

2021 ◽

Vol 13 (17) ◽

pp. 4430

Author(s):

Greet Wieme ◽

Jan Kral ◽

Toon Rosseel ◽

Petra Zemankova ◽

Bram Parton ◽

...

Keyword(s):

Ductal Adenocarcinoma ◽

Multiple Primary Cancers ◽

Cancer History ◽

Panel Testing ◽

Pathogenic Variants ◽

Increased Risk ◽

Predisposition Genes ◽

Family Cancer History ◽

Multigene Panel Testing ◽

Multigene Panel

(1) Background: The proportion and spectrum of germline pathogenic variants (PV) associated with an increased risk for pancreatic ductal adenocarcinoma (PDAC) varies among populations. (2) Methods: We analyzed 72 Belgian and 226 Czech PDAC patients by multigene panel testing. The prevalence of pathogenic variants (PV) in relation to personal/family cancer history were evaluated. PDAC risks were calculated using both gnomAD-NFE and population-matched controls. (3) Results: In 35/298 (11.7%) patients a PV in an established PDAC-predisposition gene was found. BRCA1/2 PV conferred a high risk in both populations, ATM and Lynch genes only in the Belgian subgroup. PV in other known PDAC-predisposition genes were rarer. Interestingly, a high frequency of CHEK2 PV was observed in both patient populations. PV in PDAC-predisposition genes were more frequent in patients with (i) multiple primary cancers (12/38; 32%), (ii) relatives with PDAC (15/56; 27%), (iii) relatives with breast/ovarian/colorectal cancer or melanoma (15/86; 17%) but more rare in sporadic PDAC (5/149; 3.4%). PV in homologous recombination genes were associated with improved overall survival (HR = 0.51; 95% CI 0.34–0.77). (4) Conclusions: Our analysis emphasizes the value of multigene panel testing in PDAC patients, especially in individuals with a positive family cancer history, and underlines the importance of population-matched controls for risk assessment.

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Protein haploinsufficiency drivers identify MYBPC3 mutations that cause hypertrophic cardiomyopathy

10.1101/2020.05.04.20087726 ◽

2020 ◽

Cited By ~ 2

Author(s):

Carmen Suay-Corredera ◽

Maria Rosaria Pricolo ◽

Elías Herrero-Galán ◽

Diana Velázquez-Carreras ◽

David Sánchez-Ortiz ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Protein Stability ◽

Rna Splicing ◽

Specific Gene ◽

Gene Encoding ◽

Pathogenic Variants ◽

Myosin Binding Protein ◽

Disease Mutations ◽

Uncertain Significance ◽

Molecular Phenotypes

ABSTRACTHypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Mutations in MYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), are a leading cause of HCM. However, it remains challenging to define whether specific gene variants found in patients are pathogenic or not, limiting the reach of cardiovascular genetics in the management of HCM. Here, we have examined cMyBP-C haploinsufficiency drivers in 68 clinically annotated non-truncating variants of MYBPC3. We find that 45% of the pathogenic variants show alterations in RNA splicing or protein stability, which can be linked to pathogenicity with 100% and 94% specificity, respectively. Relevant for variant annotation, we uncover that 9% of non-truncating variants of MYBPC3 currently classified as of uncertain significance induce one of these molecular phenotypes. We propose that alteration of RNA splicing or protein stability caused by MYBPC3 variants provide strong evidence of their pathogenicity, leading to improved clinical management of HCM patients and their families.

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The First Korean Case of SLC12A3 Aberrant Skipping of Two Exons Detected by RNA Splicing Analysis

Case Reports in Nephrology and Dialysis ◽

10.1159/000517139 ◽

2021 ◽

Vol 11 (2) ◽

pp. 210-213

Author(s):

Kiwoong Ko ◽

Jong-Won Kim

Keyword(s):

Rna Splicing ◽

Autosomal Recessive ◽

Gitelman Syndrome ◽

Recessive Trait ◽

Aberrant Splicing ◽

Variant Of Uncertain Significance ◽

Pathogenic Variants ◽

Exonic Variant ◽

Uncertain Significance ◽

Potential Pathogenicity

Gitelman syndrome is a salt-losing tubular disorder that is transmitted as an autosomal recessive trait. Variants in the SLC12A3 gene are found in the majority of Gitelman syndrome patients. A 26-year-old woman visited the genetic counseling clinic. Her fiancé was a known Gitelman syndrome patient who was previously diagnosed with 2 pathogenic variants in SLC12A3. In advance of marriage and future family planning, she wanted to perform genetic testing of SLC12A3. A silent exonic variant c.1050G>A was found, and multiple splice site in silico algorithms predicted this variant to have potential alteration of splicing. This variant was classified as “variant of uncertain significance,” and RNA splicing analysis was additionally performed. RNA splicing analysis showed aberrant splicing of exon 7–8 skipping. The result points out the potential pathogenicity of this variant, which should be considered a candidate of variant reclassification in the future. We highly recommend the performance of additional RNA splicing analysis, especially for silent variants predicted to have potential alteration of splicing.

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Genetic testing for breast cancer risk, from BRCA1/2 to a seven gene panel: an ethical analysis

BMC Medical Ethics ◽

10.1186/s12910-020-00545-8 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Erik Gustavsson ◽

Giovanni Galvis ◽

Niklas Juth

Keyword(s):

Breast Cancer ◽

Genetic Testing ◽

Cost Effectiveness ◽

Gene Panel ◽

Special Focus ◽

Secondary Findings ◽

Variants Of Uncertain Significance ◽

Pathogenic Variants ◽

Increased Risk ◽

Uncertain Significance

Abstract Background Genetic testing is moving from targeted investigations of monogenetic diseases to broader testing that may provide more information. For example, recent health economic studies of genetic testing for an increased risk of breast cancer suggest that it is associated with higher cost-effectiveness to screen for pathogenic variants in a seven gene panel rather than the usual two gene test for variants in BRCA1 and BRCA2. However, irrespective of the extent to which the screening of the panel is cost-effective, there may be ethical reasons to not screen for pathogenic variants in a panel, or to revise the way in which testing and disclosing of results are carried out. Main text In this paper we discuss the ethical aspects of genetic testing for an increased risk of breast cancer with a special focus on the ethical differences between screening for pathogenic variants in BRCA1/2 and a seven gene panel. The paper identifies that the panel increases the number of secondary findings as well as the number of variants of uncertain significance as two specific issues that call for ethical reflection. Conclusions We conclude that while the problem of handling secondary findings should not be overstated with regard to the panel, the fact that the panel also generate more variants of uncertain significance, give rise to a more complex set of problems that relate to the value of health as well as the value of autonomy. Therefore, it is insufficient to claim that the seven gene panel is preferable by only referring to the higher cost effectiveness of the panel.

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