Functional evaluation of variants of unknown significance in the BRCA2 gene identified in genetic testing

10002 Background: BRCA1 and BRCA2 mutations in the general population are rare. Women with these mutations have a significantly increased risk of invasive breast and ovarian cancer (65–85% and 15–65% cumulative lifetime risk, respectively). Variants of unknown significance (VUS), which are of uncertain clinical importance, account for up to 50% of all identified BRCA1 and BRCA2 sequence alterations1. Methods: Pooled data from all patients presenting to Fox Chase Cancer Center for genetic counseling was examined. Patients underwent genetic testing after detailed genetic counseling. Clinical data, including gender, ethnic background, and personal history of cancer, and total number of patients tested were collected. Results: A total of 1,765 women and 236 men underwent genetic testing. The distribution of ethnicity was: <1% Asian, 2.7% African American, <1% Hispanic, 2.4% other or of more than one ethnicity, 83% White, and 11% unknown. Mutations of BRCA1 and BRCA2 were seen in 13% of the women and 2.7% of the men. VUS were seen in 6.2% of the women and .15% of the men. Of the women positive for a VUS, 2.4% were Asian, 18.1% were African American, 5.5% were Hispanic, 4.7% were more than one ethnicity, 66.9% were White, and 2.4% were Unknown ethnicity. Only .15% of the men tested were positive for a VUS, all of whom were White. Of the 51 African American women tested, 45.1% were positive for a VUS while only 5.5% of the 1,503 White women tested were positive (p<0.0001). Of the females testing positive for a VUS, a personal history of breast cancer was seen in 66.7% of Asians, 78.3% of African Americans, 100% of Hispanics, 83.3% of those more than one race, 61% of Whites, and none of the people of unknown ethnic origin. One of three men testing positive for a VUS reported a history of breast cancer. Conclusions: Identification of VUS occurred disproportionately in African Americans, occurring ten times more often in African American women than White women in our study. Studies to improve classification of VUS as deleterious or neutral are needed to enhance the utility of genetic testing for women at risk, particularly those of African American ethnicity. 1Goldman, DE et al. Am. J. Hum. Genet., 2004. No significant financial relationships to disclose.

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Facilitations and Hurdles of Genetic Testing in Neuromuscular Disorders

Diagnostics ◽

10.3390/diagnostics11040701 ◽

2021 ◽

Vol 11 (4) ◽

pp. 701

Author(s):

Andrea Barp ◽

Lorena Mosca ◽

Valeria Ada Sansone

Keyword(s):

Genetic Testing ◽

Neuromuscular Disorders ◽

Single Nucleotide Variants ◽

Gene Therapies ◽

Variants Of Unknown Significance ◽

Unknown Significance ◽

New Gene ◽

Genetic Techniques ◽

Definition Of ◽

Next Generation Sequencing Ngs

Neuromuscular disorders (NMDs) comprise a heterogeneous group of disorders that affect about one in every thousand individuals worldwide. The vast majority of NMDs has a genetic cause, with about 600 genes already identified. Application of genetic testing in NMDs can be useful for several reasons: correct diagnostic definition of a proband, extensive familial counselling to identify subjects at risk, and prenatal diagnosis to prevent the recurrence of the disease; furthermore, identification of specific genetic mutations still remains mandatory in some cases for clinical trial enrollment where new gene therapies are now approaching. Even though genetic analysis is catching on in the neuromuscular field, pitfalls and hurdles still remain and they should be taken into account by clinicians, as for example the use of next generation sequencing (NGS) where many single nucleotide variants of “unknown significance” can emerge, complicating the correct interpretation of genotype-phenotype relationship. Finally, when all efforts in terms of molecular analysis have been carried on, a portion of patients affected by NMDs still remain “not genetically defined”. In the present review we analyze the evolution of genetic techniques, from Sanger sequencing to NGS, and we discuss “facilitations and hurdles” of genetic testing which must always be balanced by clinicians, in order to ensure a correct diagnostic definition, but taking always into account the benefit that the patient could obtain especially in terms of “therapeutic offer”.

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The underestimated burden of monogenic kidney disease in adults waitlisted for kidney transplantation

Genetics in Medicine ◽

10.1038/s41436-021-01127-8 ◽

2021 ◽

Author(s):

Eva Schrezenmeier ◽

Elisa Kremerskothen ◽

Fabian Halleck ◽

Oliver Staeck ◽

Lutz Liefeldt ◽

...

Keyword(s):

Kidney Transplantation ◽

Genetic Testing ◽

Kidney Disease ◽

Diagnostic Value ◽

Considerable Proportion ◽

Major Health ◽

Variants Of Unknown Significance ◽

Health Care Burden ◽

Unknown Significance ◽

Next Generation Sequencing Ngs

Abstract Purpose Chronic kidney disease (CKD) is a major health-care burden. Increasing evidence suggests that a considerable proportion of patients are affected by a monogenic kidney disorder. Methods In this study, the kidney transplantation waiting list at the Charité was screened for patients with undetermined cause of CKD. By next-generation sequencing (NGS) we targeted all 600 genes described and associated with kidney disease or allied disorders. Results In total, 635 patients were investigated. Of these, 245 individuals had a known cause of CKD (38.5%) of which 119 had a proven genetic disease (e.g., ADPKD, Alport). The other 340 patients (53.5%) were classified as undetermined diagnosis, of whom 87 had kidney failure (KF) onset <40 years. To this latter group genetic testing was offered as well as to those patients (n = 29) with focal segmental glomerulosclerosis (FSGS) and all individuals (n = 21) suspicious for thrombotic microangiopathy (TMA) in kidney biopsy. We detected diagnostic variants in 26 of 126 patients (20.6%) of which 14 of 126 (11.1%) were pathogenic or likely pathogenic. In another 12 of 126 (9.5%) patients, variants of unknown significance (VUS) were detected. Conclusion Our study demonstrates the diagnostic value of comprehensive genetic testing among patients with undetermined CKD.

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In silico analysis of BRCA1 and BRCA2 missense variants and the relevance in molecular genetic testing

Scientific Reports ◽

10.1038/s41598-021-88586-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kok-Siong Poon

Keyword(s):

Genetic Testing ◽

Molecular Genetic ◽

In Silico Analysis ◽

Molecular Testing ◽

Brca1 And Brca2 ◽

Missense Variants ◽

Variants Of Unknown Significance ◽

Sorting Intolerant From Tolerant ◽

Unknown Significance ◽

High Concordance

AbstractOver the years since the genetic testing of BRCA1 and BRCA2 has been conducted for research and later introduced into clinical practice, a high number of missense variants have been reported in the literature and deposited in public databases. Polymorphism Phenotyping v2 (PolyPhen-2) and Sorting Intolerant from Tolerant (SIFT) are two widely applied bioinformatics tools used to assess the functional impacts of missense variants. A total of 2605 BRCA1 and 4763 BRCA2 variants from the ClinVar database were analysed with PolyPhen2 and SIFT. When SIFT was evaluated alongside PolyPhen-2 HumDiv and HumVar, it had shown top performance in terms of negative predictive value (NPV) (100%) and sensitivity (100%) for ClinVar classified benign and pathogenic BRCA1 variants. Both SIFT and PolyPhen-2 HumDiv achieved 100% NPV and 100% sensitivity in prediction of pathogenicity of the BRCA2 variants. Agreement was achieved in prediction outcomes from the three tested approaches in 55.04% and 68.97% of the variants of unknown significance (VUS) for BRCA1 and BRCA2, respectively. The performances of PolyPhen-2 and SIFT in predicting functional impacts varied across the two genes. Due to lack of high concordance in prediction outcomes among the two tested algorithms, their usefulness in classifying the pathogenicity of VUS identified through molecular testing of BRCA1 and BRCA2 is hence limited in the clinical setting.

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High-Throughput Functional Evaluation of BRCA2 Variants of Unknown Significance

10.1101/2020.01.17.911057 ◽

2020 ◽

Author(s):

Masachika Ikegami ◽

Shinji Kohsaka ◽

Toshihide Ueno ◽

Yukihide Momozawa ◽

Kenji Tamura ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Parp Inhibitors ◽

Convincing Evidence ◽

International Agency ◽

Functional Evaluation ◽

Variants Of Unknown Significance ◽

B Method ◽

Companion Diagnostic ◽

Unknown Significance

ABSTRACTNumerous nontruncating missense variants of the BRCA2 gene have been identified, but there is a lack of convincing evidence, such as familial data, demonstrating their clinical relevance and they thus remain unactionable. To assess the pathogenicity of variants of unknown significance (VUSs) within BRCA2, we developed a novel method, the MANO-B method, for high-throughput functional evaluation utilizing BRCA2-deficient cells and poly(ADP-ribose) polymerase (PARP) inhibitors. The estimated sensitivity and specificity of this assay compared to those of the International Agency for Research on Cancer (IARC) classification system were 95% and 95%, respectively. We classified the pathogenicity of 186 BRCA2 VUSs with our original computational pipeline, resulting in the classification of 126 mutations as “neutral/likely neutral”, 23 as “intermediate”, and 37 as “deleterious/likely deleterious”. We further invented a simplified, on-demand annotation system, the Accurate BRCA Companion Diagnostic (ABCD) test, as a companion diagnostic for PARP inhibitors in patients with unknown BRCA2 VUSs. The ABCD test classification was reproducible and consistent with that of a large-scale MANO-B method.

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Genetic risk assessment for hereditary RCC: Report from the consensus panel meeting.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.6_suppl.615 ◽

2020 ◽

Vol 38 (6_suppl) ◽

pp. 615-615

Author(s):

Michael Daneshvar ◽

Neil Mendhiratta ◽

Ramaprasad Srinivasan ◽

Eric Jonasch ◽

Mark Wayne Ball ◽

...

Keyword(s):

Risk Assessment ◽

Genetic Testing ◽

Kidney Cancer ◽

Genetic Risk ◽

Genetic Counselors ◽

Genetic Risk Assessment ◽

Variants Of Unknown Significance ◽

Modified Delphi ◽

Unknown Significance ◽

Patient Advocates

615 Background: While many genes are now known to be associated with hereditary kidney cancer syndromes, there is a paucity of guidelines or uniform consensus on genetic testing for these patients. An expert panel was organized to assess who, what, when and how patients should be evaluated and what testing should be initiated. Methods: A national, multidisciplinary, panel of experts in urology, medical oncology, clinical geneticists, genetic counselors and patient advocates with background and knowledge in hereditary syndromic kidney cancer convened in person in September 2019. A renal cell carcinoma (RCC) genetic risk assessment questionnaire consisting of 52 questions was compiled prior to the meeting using modified Delphi methodology. The questions were then discussed and reviewed with uniform consensus defined as a minimum of 85% agreement in accordance with the National Comprehensive Cancer Network criteria. Results: The panel consisted of twenty-six attendees represented by urologists (43%), medical oncologist (23%), genetic counselors (13%), clinical geneticists (7%), and patient advocates (3%). The questionnaire consisted of fifty-five statements focusing on who, what, when and how genetic testing should be performed in a patient suspected of hereditary RCC syndrome. A >85% agreement was reached on 30/52 statements with 18/25 (72%) achieving consensus addressing “who”, 2/6 (33%) achieving consensus in “what’ category, 2/7 (29%) in ‘when’ and 4/6 (67%) on how. The questions with least consensus were found in the “what/when?” category with only 4/13 questions with minimum 85% agreement. Specific areas of debate included an age cutoff for prompting a genetic risk assessment as well as need for familial testing in patients with variants of unknown significance. Conclusions: Despite experience of the panel in management of hereditary RCC, the consensus was reached only on 66% of genetic testing. While many issues will need to be discussed further, those statements with consensus may be used to guide physicians and patients on who, what, when and how genetic RCC risk assessment should be performed.

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Ethical Considerations in Neurogenetic Testing

Seminars in Neurology ◽

10.1055/s-0038-1667382 ◽

2018 ◽

Vol 38 (05) ◽

pp. 505-514 ◽

Cited By ~ 2

Author(s):

Xiaowei Su ◽

Zachary Simmons

Keyword(s):

Genetic Testing ◽

Ethical Issues ◽

Diagnostic Testing ◽

Ethical Principles ◽

Ethical Framework ◽

Complex Nature ◽

Neurologic Diseases ◽

Variants Of Unknown Significance ◽

Unknown Significance ◽

The One

AbstractRecent advances in the genetics of neurologic diseases coupled with improvements in sensitivity and specificity are making genetic testing an increasingly important part of diagnosis and management for neurologists. However, the complex nature of genetic testing, the nuances of multiple result types, and the short- and long-term consequences of genetic diagnoses raise important ethical issues for the clinician. Neurologists must balance the ethical principles of beneficence and nonmaleficence, on the one hand, with patient autonomy on the other hand, when ordering such tests by facilitating shared decision making, carrying out their fiduciary responsibilities to patients, and ensuring that patients have adequate counseling to make informed decisions. This review summarizes ethical issues related to genetic testing for neurologic diseases, with a focus on clinical practice. Informed consent for genetic testing of patients and asymptomatic at-risk family members is discussed. The roles and responsibilities of physicians as genetic counselors are reviewed, including the framing of incidental findings and variants of unknown significance that impact individuals' decisions about whether to pursue genetic testing and what results they wish to know. Disclosure and its consequences for the patient are placed within an ethical framework to permit a better understanding of why genetic testing is different from most other diagnostic testing ordered by physicians. The review ends with clinical vignettes that attempt to place ethical principles into familiar clinical settings involving physicians, patients and their families.

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High Throughput Functional Evaluation of KCNQ1 Decrypts Variants of Unknown Significance

10.1101/223206 ◽

2017 ◽

Cited By ~ 1

Author(s):

Carlos G. Vanoye ◽

Reshma R. Desai ◽

Katarina L. Fabre ◽

Franck Potet ◽

Jean-Marc DeKeyser ◽

...

Keyword(s):

High Throughput ◽

Genetic Diseases ◽

Functional Evaluation ◽

Loss Of Function ◽

Gene Variation ◽

Variants Of Unknown Significance ◽

Cardiac Ion Channels ◽

Unknown Significance ◽

High Level

ABSTRACTBackgroundThe explosive growth in known human gene variation presents enormous challenges to current approaches for variant classification that impact diagnosis and treatment of many genetic diseases. For disorders caused by mutations in cardiac ion channels, such as congenital long-QT syndrome (LQTS), in vitro electrophysiological evidence has high value in discriminating pathogenic from benign variants, but these data are often lacking because assays are cost-, time- and labor-intensive.Methods and ResultsWe implemented a strategy for performing high throughput, functional evaluations of ion channel variants that repurposed an automated electrophysiology platform developed previously for drug discovery. We demonstrated success of this approach by evaluating 78 variants in KCNQ1, a major LQTS gene. We benchmarked our results with traditional electrophysiological approaches and observed a high level of concordance. Our results provided functional data useful for classifying ~70% of previously unstudied KCNQ1 variants annotated with uninformative descriptions in the public database ClinVar. Further, we show that rare and ultra-rare KCNQ1 variants in the general population exhibit functional properties ranging from normal to severe loss-of-function indicating that allele frequency is not a reliable predictor of channel function.ConclusionsOur results illustrate an efficient and high throughput paradigm linking genotype to function for a human cardiac channelopathy that will enable data-driven classification of large numbers of variants and create new opportunities for precision medicine.

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P3567Genetic screening for monogenic hypertension in hypertensive individuals in a clinical setting

European Heart Journal ◽

10.1093/eurheartj/ehz745.0429 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

M Bao ◽

J Zhong ◽

J Cai ◽

X Yang

Keyword(s):

Functional Analysis ◽

Diagnostic Yield ◽

Basic Research ◽

Functional Evaluation ◽

Variants Of Unknown Significance ◽

Treatment And Prevention ◽

Pathogenic Variants ◽

Unknown Significance ◽

Monogenic Hypertension ◽

Number Of Individuals

Abstract Objectives Monogenic hypertension describes a series of hypertension syndromes inherited by Mendelian law and present with complex phenotypes. Methods 1179 cases with monogenic hypertension potential were evaluated by sequencing 37 causative genes. Pathogenic variants were classified by using American College of Medical Genetics guidelines. Additionally, 49 variants of unknown significance were selected to receive functional analysis. The yield of combined genetic and functional analysis was evaluated. Results 21 deleterious variants were identified in 33 of 1179 (2.80%). Functional analysis for 49 unknown significant variants showed 32 variants harbored by 61 individuals led to abnormally expressed protein levels. Overall, combining genetic screening with functional analysis promoted diagnostic yield to 8.73%. The main etiology established was primary aldosteronism, with CACNA1H harboring the greatest mutation burden. Logistic regression analysis showed hypertension complicated with special manifestations had the strongest correlation with disease causing variants detection (p=0.03). Sequencing Results Summary Number of variants Number of individuals* Percentage† Individuals with no variant 0 524 44.44% Individuals with variants identified 592 655 55.56% Individuals with single contributing variant 297 480 40.71% Individuals with two or multiple contributing variants 295 175 14.84% Number of variants identified Pathogenic and likely pathogenic variants 21 33 2.80% Variants of unknown significance 570 634 53.77% Benign or likely benign variants 1 1 0.08% Type of variant Frameshift deletion 8 15 1.27% Frameshift insertion 5 5 0.42% Nonframeshift deletion 10 10 0.85% Nonframeshift insertion 6 12 1.02% Nonsynonymous SNV 546 607 51.48% Stopgain SNV 18 30 2.54% WES, whole-exome sequencing. *The statistics in this table was based on 1179 individuals. †The percentage was calculated by the number of individuals in each category. A flow chart of this study. Conclusion Our findings demonstrate an enhanced diagnostic ability by combining genetic analysis with functional evaluation and enables targeted treatment and prevention of hypertension. Acknowledgement/Funding This work was supported by National Basic Research Program of China (973 Program, 2014CB542300, 2014CB542302).

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