Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance

Woods, Nicholas T.; Baskin, Rebekah; Golubeva, Volha A.; Jhuraney, Ankita; De-Gregoriis, Giuliana; Vaclová, Tereza; Goldgar, David E.; Couch, Fergus J.; Carvalho, Marcelo A.; Iversen, Edwin S.; Monteiro, Alvaro N.A.

doi:10.1038/npjgenmed.2016.1

Cited by 77 publications

(86 citation statements)

References 44 publications

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“…In the following analysis, we adopted the Bayes factor (BF) as the strength of evidence in favor of pathogenicity. To calculate the BF, we utilized a Bayesian hierarchical two-component Gaussian mixture model based on the VarCall model 18,31,32 with a noninformative prior probability. In this setting, the BFs were calculated as the probability ratio of a variant being deleterious to it being neutral.…”

Section: Resultsmentioning

confidence: 99%

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

Ikegami

Ueno

Momozawa

et al. 2020

Preprint

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Keywords: BRCA2, poly(ADP-ribose) polymerase (PARP) inhibitor, variants of unknown 22 significance (VUSs), companion diagnosis, Bayesian hierarchical model 23 24 ABSTRACT 44Numerous nontruncating missense variants of the BRCA2 gene have been identified, but there 45 is a lack of convincing evidence, such as familial data, demonstrating their clinical relevance 46 and they thus remain unactionable. To assess the pathogenicity of variants of unknown 47The BRCA1 and BRCA2 (BRCA) genes encode key proteins in the homology-directed DNA 60 break repair (HDR) pathway, and their inactivation predisposes individuals to cancer 61 development 1 . Germline loss-of-function variants in BRCA markedly increase the risk of early-62 onset breast and ovarian cancer; in such cases, prophylactic oophorectomy and mastectomy 63 and genetic testing for at-risk relatives must be considered 2, 3, 4 . Tumors with pathogenic 64 mutations within BRCA and defective HDR have been shown to be particularly sensitive to 65 platinum-based chemotherapies and PARP inhibitors, the efficacy of which is mediated 66 through synthetic lethality in cancer cells with BRCA loss-of-function 5, 6 . 67 68The American College of Medical Genetics and Genomics (ACMG) standards and guidelines 69 for the interpretation of sequence variants recommend a process for variant classification based 70 on criteria using population, computational, functional, and segregation data 7 . Family-based 71 studies including a multifactorial model of pathology, a cosegregation profile, and the 72 cooccurrence and family history of cancer may exemplify the most reliable methods for 73 classifying BRCA2 gene variants 8, 9 . Nonsense or frameshift mutations within the coding exons 74 of BRCA markedly alter the structures of the protein products and are presumed to confer loss-75 of-function. However, the vast majority of missense variants are individually rare in both 76 general populations and cancer patients, and case-control studies may not have sufficient 77 statistical significance to classify these variants as pathogenic or benign 10, 11, 12 . No current in 78 silico computational prediction algorithm for missense variants is accurate enough when used 79 alone 13 . Thus, the functional evaluation of missense variants of unknown significance (VUSs) 80 is urgently required to improve the interpretation of variants identified by genetic testing and 81 to support clinical decision-making for their carriers 14 . 82Page 5 of 63 While thousands of BRCA1 VUSs have been assessed by recently developed high-throughput 83 functional assays 15, 16, 17 , a few hundred BRCA2 variants have been evaluated by a conventional 84 functional assay for BRCA2, the HDR assay 18, 19 . The HDR assay has three issues requiring 85 improvement: (i) the throughput is quite low, (ii) it uses a hamster cell line with transient 86 expression of BRCA2 cDNA, and most importantly, (iii) it can evaluate only variants in the 87 DNA-binding domain 14, 20 . To overcome these limitations, we propose herein a high-8...

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Section: Resultsmentioning

confidence: 99%

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

Ikegami

Ueno

Momozawa

et al. 2020

Preprint

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“…A similar dilemma is faced in annotation of BRCA2 (MIM: 600185) variants in which a probability threshold and annotation for deleterious variants associated with only moderate risk for cancer have yet to be determined. 58 The ongoing challenge of annotation of variants with moderate functional effects lends itself well to the incorporation of quantitative functional results and their associated endophenotypes (continuous-valued quantitative traits) 59 into a variant classification scheme.…”

Section: Variant Countmentioning

confidence: 99%

Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity

Raraigh

Han

Davis

et al. 2018

The American Journal of Human Genetics

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Missense DNA variants have variable effects upon protein function. Consequently, interpreting their pathogenicity is challenging, especially when they are associated with disease variability. To determine the degree to which functional assays inform interpretation, we analyzed 48 CFTR missense variants associated with variable expressivity of cystic fibrosis (CF). We assessed function in a native isogenic context by evaluating CFTR mutants that were stably expressed in the genome of a human airway cell line devoid of endogenous CFTR expression. 21 of 29 variants associated with full expressivity of the CF phenotype generated <10% wild-type CFTR (WT-CFTR) function, a conservative threshold for the development of life-limiting CF lung disease, and five variants had moderately decreased function (10% to ∼25% WT-CFTR). The remaining three variants in this group unexpectedly had >25% WT-CFTR function; two were higher than 75% WT-CFTR. As expected, 14 of 19 variants associated with partial expressivity of CF had >25% WT-CFTR function; however, four had minimal to no effect on CFTR function (>75% WT-CFTR). Thus, 6 of 48 (13%) missense variants believed to be disease causing did not alter CFTR function. Functional studies substantially refined pathogenicity assignment with expert annotation and criteria from the American College of Medical Genetics and Genomics and Association for Molecular Pathology. However, four algorithms (CADD, REVEL, SIFT, and PolyPhen-2) could not differentiate between variants that caused severe, moderate, or minimal reduction in function. In the setting of variable expressivity, these results indicate that functional assays are essential for accurate interpretation of missense variants and that current prediction tools should be used with caution.

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“…The assay was further validated by the functional impact of known cancer-associated variants and BRCA1 TA. Over the last 15 years, more than 300 variants enclosed within the 1396-1863 amino acid residues (all known missense variants in the C-terminal region of the BRCA1) were evaluated [57,58]. By comparing the results of BRCA1 TA with a reference panel of 49 missense variants classified by multifactorial models, using a Bayesian hierarchical model (VarCall) to predict the likelihood of pathogenicity, we observed a 1.0 sensitivity and 1.0 specificity (lower bound of 95% confidence interval = 0.78 and 0.84, respectively), demonstrating that this approach can classify VUS with confidence [57].…”

Section: Functional Assaysmentioning

confidence: 99%

Germline Missense Variants in BRCA1: New Trends and Challenges for Clinical Annotation

Golubeva¹,

Nepomuceno²,

Monteiro³

2019

Preprint

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Genetic testing allows for identification of germline DNA variations which are associated with a significant increase in risk of developing breast and ovarian cancer. Detection of a BRCA1 or BRCA2 pathogenic variant triggers several clinical management actions, which may include increased surveillance and prophylactic surgery for healthy carriers or treatment with PARP inhibitor therapy for carriers diagnosed with cancer. Thus, standardized validated criteria for annotation of BRCA1 and BRCA2 variants according to their pathogenicity are necessary to support clinical decision making and ensure improved outcomes. Upon detection, variants whose pathogenicity can be inferred by the genetic code are typically classified as pathogenic, likely pathogenic, likely benign, or benign. Variants whose impact on function cannot be directly inferred by the genetic code are labeled as Variants of Uncertain Clinical Significance (VUS) and are evaluated by multifactorial likelihood models that use personal and family history of cancer, segregation data, prediction tools, and co-occurrence with a pathogenic BRCA variant. Missense variants, coding alterations that replace a single amino acid residue with another, are a class of variants for which determination of clinical relevance is particularly challenging. Here, we discuss current issues in variant classification by following a typical life cycle of a BRCA1 missense variant through detection, annotation and information dissemination. Advances in massively parallel sequencing have led to a substantial increase in VUS findings. Although comprehensive assessment and classification of missense variants according to their pathogenicity remains the bottle neck, new developments in functional analysis, high throughput assays, data sharing, and statistical models are rapidly changing this scenario.

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Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance

Cited by 77 publications

References 44 publications

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity

Germline Missense Variants in BRCA1: New Trends and Challenges for Clinical Annotation

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