variants of unknown significance
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2022 ◽  
pp. practneurol-2021-002989
Author(s):  
Thanuja Dharmadasa ◽  
Jakub Scaber ◽  
Evan Edmond ◽  
Rachael Marsden ◽  
Alexander Thompson ◽  
...  

A minority (10%–15%) of cases of amyotrophic lateral sclerosis (ALS), the most common form of motor neurone disease (MND), are currently attributable to pathological variants in a single identifiable gene. With the emergence of new therapies targeting specific genetic subtypes of ALS, there is an increasing role for routine genetic testing for all those with a definite diagnosis. However, potential harm to both affected individuals and particularly to asymptomatic relatives can arise from the indiscriminate use of genetic screening, not least because of uncertainties around incomplete penetrance and variants of unknown significance. The most common hereditary cause of ALS, an intronic hexanucleotide repeat expansion in C9ORF72, may be associated with frontotemporal dementia independently within the same pedigree. The boundary of what constitutes a possible family history of MND has therefore extended to include dementia and associated psychiatric presentations. Notwithstanding the important role of clinical genetics specialists, all neurologists need a basic understanding of the current place of genetic testing in MND, which holds lessons for other neurological disorders.


2022 ◽  
Author(s):  
Willow Coyote-Maestas ◽  
David Nedrud ◽  
Yungui He ◽  
Daniel Schmidt

A longstanding goal in protein science and clinical genetics is to develop quantitative models of sequence, structure, and function relationships and delineate the mechanisms by which mutations cause disease. Deep Mutational Scanning (DMS) is a promising strategy to map how amino acids contribute to protein structure and function and to advance clinical variant interpretation. Here, we introduce 7,429 single residue missense mutation into the Inward Rectifier K+ channel Kir2.1 and determine how this affects folding, assembly, and trafficking, as well as regulation by allosteric ligands and ion conduction. Our data provide high-resolution information on a cotranslationally-folded biogenic unit, trafficking and quality control signals, and segregated roles of different structural elements in fold-stability and function. We show that Kir2.1 trafficking mutants are underrepresented in variant effect databases, which has implications for clinical practice. By comparing fitness scores with expert-reviewed variant effects, we can predict the pathogenicity of variants of unknown significance and disease mechanisms of know pathogenic mutations. Our study in Kir2.1 provides a blueprint for how multiparametric DMS can help us understand the mechanistic basis of genetic disorders and the structure-function relationships of proteins.


2021 ◽  
Vol 8 ◽  
Author(s):  
Susan E. Tsutakawa ◽  
Albino Bacolla ◽  
Panagiotis Katsonis ◽  
Amer Bralić ◽  
Samir M. Hamdan ◽  
...  

All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase–nuclease–RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary “driver” and “passenger” mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.


Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1951
Author(s):  
Katarzyna Aleksandra Jalowiec ◽  
Kristina Vrotniakaite-Bajerciene ◽  
Annina Capraru ◽  
Tatiana Wojtovicova ◽  
Raphael Joncourt ◽  
...  

(1) Background: Clinical and molecular data on patients with unexplained erythrocyto-sis is sparse. We aimed to analyze the clinical and molecular features of patients with congenital erythrocytosis in our tertiary reference center. (2) Methods: In 34 patients with unexplained erythrocytosis, a 13-gene Next-Generation Sequencing erythrocytosis panel developed at our center was conducted. (3) Results: In 6/34 (18%) patients, eight different heterozygous gene variants were found. These patients were, therefore, diagnosed with congenital erythrocytosis. Two patients had two different gene variants each. All variants were characterized as variants of unknown significance as they had not previously been described in the literature. The rest of the patients (28/34, 82%) had no detected gene variants. (4) Conclusions: Our experience shows that the NGS panel can be helpful in determining the reasons for persistent, unexplained erythrocytosis. In our cohort of patients with erythrocytosis, we identified some, thus far unknown, gene variants which may explain the clinical picture. However, further investigations are needed to determine the relationship between the molecular findings and the phenotype.


2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Kai Yu Ma ◽  
Michiel R. Fokkens ◽  
Teus van Laar ◽  
Dineke S. Verbeek

AbstractPathogenic variants in PINK1 cause early-onset Parkinson’s disease. Although many PINK1 variants have been reported, the clinical significance is uncertain for the majority of them. To gain insights into the consequences of PINK1 missense variants in a systematic manner, we selected 50 PINK1 missense variants from patient- and population-wide databases and systematically classified them using Sherloc, a comprehensive framework for variant interpretation based on ACMG-AMP guidelines. We then performed functional experiments, including mitophagy and Parkin recruitment assays, to assess the downstream consequences of PINK1 variants. Analysis of PINK1 missense variants based on Sherloc showed that the patient databases over-annotate variants as likely pathogenic. Furthermore, our study shows that pathogenic PINK1 variants are most often linked to a loss-of-function for mitophagy and Parkin recruitment, while this is not observed for variants of unknown significance. In addition to the Sherloc framework, the added layer of evidence of our functional tests suggests a reclassification of 9/50 missense variants. In conclusion, we suggest the assessment of multiple layers of evidence, including functional data on top of available clinical and population-based data, to support the clinical classification of a variant and show that the presence of a missense variant in PINK1 in a Parkinson’s disease case does not automatically imply pathogenicity.


2021 ◽  
Vol 17 (S5) ◽  
Author(s):  
Bhagyashri Burgute ◽  
Alan E. Renton ◽  
Ellen Ziegemeier ◽  
Carlos Cruchaga ◽  
Alison M. Goate ◽  
...  

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1930
Author(s):  
Stephanie I. W. van de Stadt ◽  
Petra A. W. Mooyer ◽  
Inge M. E. Dijkstra ◽  
Conny J. M. Dekker ◽  
Divya Vats ◽  
...  

Due to newborn screening for X-linked adrenoleukodystrophy (ALD), and the use of exome sequencing in clinical practice, the detection of variants of unknown significance (VUS) in the ABCD1 gene is increasing. In these cases, functional tests in fibroblasts may help to classify a variant as (likely) benign or pathogenic. We sought to establish reference ranges for these tests in ALD patients and control subjects with the aim of helping to determine the pathogenicity of VUS in ABCD1. Fibroblasts from 36 male patients with confirmed ALD, 26 healthy control subjects and 17 individuals without a family history of ALD, all with an uncertain clinical diagnosis and a VUS identified in ABCD1, were included. We performed a combination of tests: (i) a test for very-long-chain fatty acids (VLCFA) levels, (ii) a D3-C22:0 loading test to study the VLCFA metabolism and (iii) immunoblotting for ALD protein. All ALD patient fibroblasts had elevated VLCFA levels and a reduced peroxisomal ß-oxidation capacity (as measured by the D3-C16:0/D3-C22:0 ratio in the D3-C22:0 loading test) compared to the control subjects. Of the VUS cases, the VLCFA metabolism was not significantly impaired (most test results were within the reference range) in 6/17, the VLCFA metabolism was significantly impaired (most test results were within/near the ALD range) in 9/17 and a definite conclusion could not be drawn in 2/17 of the cases. Biochemical studies in fibroblasts provided clearly defined reference and disease ranges for the VLCFA metabolism. In 15/17 (88%) VUS we were able to classify the variant as being likely benign or pathogenic. This is of great clinical importance as new variants will be detected.


Author(s):  
Emily Rayner ◽  
Yvonne Tiersma ◽  
Cristina Fortuno ◽  
Sandrine van Hees-Stuivenberg ◽  
Mark Drost ◽  
...  

The large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low-penetrance gene for the cancer predisposition Lynch Syndrome (LS, OMIM 120435), represent variants of unknown significance (VUS). The inability to assess pathogenicity of such VUS interferes with personalized healthcare. The complete in vitro MMR activity (CIMRA) assay, that only requires sequence information on the VUS, provides a functional analysis-based tool suited for VUS classification. To derive a formula that translates CIMRA assay results for PMS2 VUS into the odds of pathogenicity (OddsPath), we used a set of clinically classified PMS2 variants, supplemented by inactivating variants generated by an in cellulo genetic screen, as proxies for pathogenic variants. Validation of this OddsPath revealed very high predictive values for PMS2 VUS. We conclude that this OddsPath provides an integral metric that, similar to the other, higher penetrance, MMR proteins MSH2, MLH1 and MSH6, can be incorporated into the upcoming criteria for MMR gene VUS classification of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). This will represent a seminal step forward in enabling personalized healthcare for individuals suspected of LS and their relatives.


2021 ◽  
Author(s):  
Juan C Caicedo ◽  
John Arevalo ◽  
Federica Piccioni ◽  
Mark-Anthony Bray ◽  
Cathy L Hartland ◽  
...  

Most variants in most genes across most organisms have an unknown impact on the function of the corresponding gene. This gap in knowledge is especially acute in cancer, where clinical sequencing of tumors now routinely reveals patient-specific variants whose functional impact on the corresponding gene is unknown, impeding clinical utility. Transcriptional profiling was able to systematically distinguish these variants of unknown significance (VUS) as impactful vs. neutral in an approach called expression-based variant-impact phenotyping (eVIP). We profiled a set of lung adenocarcinoma-associated somatic variants using Cell Painting, a morphological profiling assay that captures features of cells based on microscopy using six stains of cell and organelle components. Using deep-learning-extracted features from each cell's image, we found that cell morphological profiling (cmVIP) can predict variants' functional impact and, particularly at the single-cell level, reveals biological insights into variants which can be explored in our public online portal. Given its low cost, convenient implementation, and single-cell resolution, cmVIP profiling therefore seems promising as an avenue for using non-gene-specific assays to systematically assess the impact of variants, including disease-associated alleles, on gene function.


2021 ◽  
Vol 12 ◽  
Author(s):  
Anna E. Patrick ◽  
Eden M. Lyons ◽  
Lisa Ishii ◽  
Alan S. Boyd ◽  
Joseph M. Choi ◽  
...  

Neonatal multisystem onset inflammatory disorder (NOMID) is a severe autoinflammatory syndrome that can have an initial presentation as infantile urticaria. Thus, an immediate recognition of the clinical symptoms is essential for obtaining a genetic diagnosis and initiation of early therapies to prevent morbidity and mortality. Herein, we describe a neonate presenting with urticaria and systemic inflammation within hours after birth who developed arthropathy and neurologic findings. Pathologic evaluation of the skin revealed an infiltration of lymphocytes, eosinophils, and scattered neutrophils. Genetic analysis identified a novel heterozygous germline variant of unknown significance in the NLRP3 gene, causing the missense mutation M408T. Variants of unknown significance are common in genetic sequencing studies and are diagnostically challenging. Functional studies of the M408T variant demonstrated enhanced formation and activity of the NLRP3 inflammasome, with increased cleavage of the inflammatory cytokine IL-1β. Upon initiation of IL-1 pathway blockade, the infant had a robust response and improvement in clinical and laboratory findings. Our experimental data support that this novel variant in NLRP3 is causal for this infant’s diagnosis of NOMID. Rapid assessment of infantile urticaria with biopsy and genetic diagnosis led to early recognition and targeted anti-cytokine therapy. This observation expands the NOMID-causing variants in NLRP3 and underscores the role of genetic sequencing in rapidly identifying and treating autoinflammatory disease in infants. In addition, these findings highlight the importance of establishing the functional impact of variants of unknown significance, and the impact this knowledge may have on therapeutic decision making.


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