A Multiplex Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function

Starita, Lea M.; Islam, Muhtadi M.; Banerjee, Tapahsama; Adamovich, Aleksandra I.; Gullingsrud, Justin; Fields, Stanley; Shendure, Jay; Parvin, Jeffrey D.

doi:10.1016/j.ajhg.2018.07.016

Cited by 98 publications

(121 citation statements)

References 36 publications

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“…Recently, two saturation mutagenesis-based high-throughput approaches evaluated the impact of 1,056 and 3,893 BRCA1 variants in exons 2-7 and 15-24, respectively [59,60]. Each study used a distinct biological role of BRCA1.…”

Section: Functional Assaysmentioning

confidence: 99%

“…Each study used a distinct biological role of BRCA1. Starita and colleagues [61] interrogated the impact of alteration on the amino-terminus of BRCA1 on its role on Homology-directed Repair (HDR) while Findlay and colleagues [59] based their assay on the requirement of a functional BRCA1 for viability in a near-haploid human cell line (HAP1). These studies demonstrated the feasibility of this approach and reported an excellent agreement with the available clinical data in ClinVar database [59].…”

Section: Functional Assaysmentioning

confidence: 99%

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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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Section: Functional Assaysmentioning

confidence: 99%

Section: Functional Assaysmentioning

confidence: 99%

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

Golubeva¹,

Nepomuceno²,

Monteiro³

2019

Preprint

View full text Add to dashboard Cite

show abstract

“…While thousands of BRCA1 VUSs have been assessed by recently developed high-throughput functional assays 15,16,17 , a few hundred BRCA2 variants have been evaluated by a conventional functional assay for BRCA2 , the HDR assay 18,19 . The HDR assay has three issues requiring improvement: (i) the throughput is quite low, (ii) it uses a hamster cell line with transient expression of BRCA2 cDNA, and most importantly, (iii) it can evaluate only variants in the DNA-binding domain 14,20 .…”

Section: Introductionmentioning

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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“…The landscape of functionally characterized variants in BRCA1 has dramatically increased because of three major analyses. Starita et al 2 , measured the impact of 1056 N-terminal variants on the homologous recombination DNA repair activity of BRCA1. Findlay et al 3 , exploited the essentiality of BRCA1 for cell survival by used a saturating genome editing approach in HAP1 cells to evaluate nearly 4,000 SNVs (n=1837 distinct missense).…”

Section: Introductionmentioning

confidence: 99%

Prediction of the functional impact of missense variants in BRCA1 and BRCA2 with BRCA-ML

Hart

Polley

Shimelis

et al. 2019

Preprint

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AbstractIn silico predictions of missense variants is an important consideration when interpreting variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes. We trained and evaluated hundreds of machine learning algorithms based on results from validated functional assays to better predict missense variants in these genes as damaging or neutral. This new optimal “BRCA-ML” model yielded a substantially more accurate method than current algorithms for interpreting the functional impact of variants in these genes, making BRCA-ML a valuable addition to data sources for VUS classification.

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A Multiplex Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function

Cited by 98 publications

References 36 publications

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

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

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

Prediction of the functional impact of missense variants in BRCA1 and BRCA2 with BRCA-ML

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