scholarly journals High Throughput Functional Evaluation of KCNQ1 Decrypts Variants of Unknown Significance

2017 ◽  
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.

Abstract 921: High-throughput functional evaluation of variants of unknown significance inERBB2

2018 ◽  
Author(s):  
Masaaki Nagano ◽  
Shinji Kohsaka ◽  
Toshihide Ueno ◽  
Shinya Kojima ◽  
Masachika Ikegami ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Evaluation ◽  
Variants Of Unknown Significance ◽  
Unknown Significance

scholarly journals Novel frameshift variant in MYL2 reveals molecular differences between dominant and recessive forms of hypertrophic cardiomyopathy

2019 ◽  
Author(s):  
Sathiya N. Manivannan ◽  
Sihem Darouich ◽  
Aida Masmoudi ◽  
David Gordon ◽  
Gloria Zender ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Exome Sequencing ◽  
Rapid Screening ◽  
Functional Study ◽  
Ventricular Muscle ◽  
Loss Of Function ◽  
Variants Of Unknown Significance ◽  
Unknown Significance ◽  
The Impact

AbstractHypertrophic cardiomyopathy (HCM) is characterized by enlargement of the ventricular muscle without dilation and is often associated with dominant pathogenic variants in cardiac sarcomeric protein genes. Here, we report a family with two infants diagnosed with infantile-onset HCM and mitral valve dysplasia that led to death before one year of age. Using exome sequencing, we discovered that one of the affected children had a homozygous frameshift variant in Myosin light chain 2 (MYL2:NM_000432.3:c.431_432delCT: p.Pro144Argfs*57;MYL2-fs), which alters the last 20 amino acids of the protein and is predicted to impact the C-terminal EF-hand (CEF) domain. The parents are unaffected heterozygous carriers of the variant and the variant is absent in control cohorts from gnomAD. The absence of the phenotype in carriers and infantile presentation of severe HCM is in contrast to HCM associated with dominant MYL2 variants. Immunohistochemical analysis of the ventricular muscle of the deceased patient with the MYL2-fs variant showed marked reduction of MYL2 expression compared to an unaffected control. In vitro overexpression studies further indicate that the MYL2-fs variant is actively degraded. In contrast, an HCM-associated missense variant (MYL2:p.Gly162Arg) and three other MYL2 stopgain variants that lead to loss of the CEF domain are stably expressed. However, stopgain variants show impaired localization suggesting a functional role for the CEF domain. The degradation of the MYL2-fs can be rescued by inhibiting the cell’s proteasome function supporting a post-translational effect of the variant. In vivo rescue experiments with a Drosophila MYL2-homolog (Mlc2) knockdown model indicate that neither MYL2-fs nor MYL2:p.Gly162Arg supports regular cardiac function. The tools that we have generated provide a rapid screening platform for functional assessment of variants of unknown significance in MYL2. Our study supports an autosomal recessive model of inheritance for MYL2 loss-of-function variants and highlights the variant-specific molecular differences found in MYL2-associated cardiomyopathies.Author SummaryWe report a novel frameshift variant in MYL2 that is associated with a severe form of infantile-onset hypertrophic cardiomyopathy. The impact of the variant is only observed in the recessive form of the disease in the proband and not in the parents who are carriers of the variant. This is in contrast to other dominant variants in MYL2 that are associated with cardiomyopathies. We compared the stability of this variant to that of other cardiomyopathy associated MYL2 variants and found molecular differences in the disease pathology. We also show different protein domain requirement for stability and localization of MYL2 in cardiomyocytes. Further, we used a fly model to demonstrate functional deficits due to the variant in the developing heart. Overall, our study shows a molecular mechanism by which loss-of-function variants in MYL2 are recessive while missense variants are dominant. We highlight the use of exome sequencing and functional testing to assist in the diagnosis of rare forms of diseases where pathogenicity of the variant is not obvious. The new tools we developed for in vitro functional study and the fly fluorescent reporter analysis will permit rapid analysis of MYL2 variants of unknown significance.

scholarly journals High-Throughput Functional Evaluation of BRCA2 Variants of Unknown Significance

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.

scholarly journals High-throughput functional evaluation of BRCA2 variants of unknown significance

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Masachika Ikegami ◽  
Shinji Kohsaka ◽  
Toshihide Ueno ◽  
Yukihide Momozawa ◽  
Satoshi Inoue ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Evaluation ◽  
Variants Of Unknown Significance ◽  
Unknown Significance

High-Throughput Functional Evaluation of Variants of Unknown Significance in ERBB2

2018 ◽  
Vol 24 (20) ◽  
pp. 5112-5122 ◽  
Author(s):  
Masaaki Nagano ◽  
Shinji Kohsaka ◽  
Toshihide Ueno ◽  
Shinya Kojima ◽  
Kanju Saka ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Evaluation ◽  
Variants Of Unknown Significance ◽  
Unknown Significance

scholarly journals High-Throughput Functional Evaluation of KCNQ1 Decrypts Variants of Unknown Significance

2018 ◽  
Vol 11 (11) ◽  
Author(s):  
Carlos G. Vanoye ◽  
Reshma R. Desai ◽  
Katarina L. Fabre ◽  
Shannon L. Gallagher ◽  
Franck Potet ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Evaluation ◽  
Variants Of Unknown Significance ◽  
Unknown Significance

Abstract 516: High-throughput functional evaluation of variants of unknown significance inEGFR

2017 ◽  
Author(s):  
Shinji Kohsaka ◽  
Masaaki Nagano ◽  
Toshihide Ueno ◽  
Yoshiyuki Suehara ◽  
Takuo Hayashi ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Evaluation ◽  
Variants Of Unknown Significance ◽  
Unknown Significance

A method of high-throughput functional evaluation of EGFR gene variants of unknown significance in cancer

2017 ◽  
Vol 9 (416) ◽  
pp. eaan6566 ◽  
Author(s):  
Shinji Kohsaka ◽  
Masaaki Nagano ◽  
Toshihide Ueno ◽  
Yoshiyuki Suehara ◽  
Takuo Hayashi ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Evaluation ◽  
Gene Variants ◽  
Egfr Gene ◽  
Variants Of Unknown Significance ◽  
Unknown Significance

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals LINC00680 enhances hepatocellular carcinoma stemness behavior and chemoresistance by sponging miR-568 to upregulate AKT3

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Gege Shu ◽  
Huizhao Su ◽  
Zhiqian Wang ◽  
Shihui Lai ◽  
Yan Wang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Mechanism Study ◽  
Downstream Signaling ◽  
Mouse Xenograft Model ◽  
High Level

Abstract Background Hepatocellular carcinoma (HCC) has an extremely poor prognosis due to the development of chemoresistance, coupled with inherently increased stemness properties. Long non-coding RNAs (LncRNAs) are key regulators for tumor cell stemness and chemosensitivity. Currently the relevance between LINC00680 and tumor progression was still largely unknown, with only one study showing its significance in glioblastoma. The study herein was aimed at identifying the role of LINC00680 in the regulation HCC stemness and chemosensitivity. Methods QRT-PCR was used to detect the expression of LINC00680, miR-568 and AKT3 in tissue specimen and cell lines. Gain- or loss-of function assays were applied to access the function of LINC00680 in HCC cells, including cell proliferation and stemness properties. HCC stemness and chemosensitivity were determined by sphere formation, cell viability and colony formation. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays were performed to examine the interaction between LINC00680 and miR-568 as well as that between miR-568 and AKT3. A nude mouse xenograft model was established for the in vivo study. Results We found that LINC00680 was remarkably upregulated in HCC tissues. Patients with high level of LINC00680 had poorer prognosis. LINC00680 overexpression significantly enhanced HCC cell stemness and decreased in vitro and in vivo chemosensitivity to 5-fluorouracil (5-Fu), whereas LINC00680 knockdown led to opposite results. Mechanism study revealed that LINC00680 regulated HCC stemness and chemosensitivity through sponging miR-568, thereby expediting the expression of AKT3, which further activated its downstream signaling molecules, including mTOR, elF4EBP1, and p70S6K. Conclusion LINC00680 promotes HCC stemness properties and decreases chemosensitivity through sponging miR-568 to activate AKT3, suggesting that LINC00680 might be a potentially important HCC diagnosis marker and therapeutic target.

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