Computational and biophysical determination of pathogenicity of variants of unknown significance in cardiac thin filament.
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ABSTRACT:
SUBMITTER: Mason AB
PROVIDER: S-EPMC8675185 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle metabolism characterized by generalized muscle rigidity, increased body temperature, rhabdomyolysis, hyperkalemia and severe metabolic acidosis. The underlying mechanism of MH involves excessive Ca2+ release from myotubes via the ryanodine receptor type 1 (RYR1) and the voltage-dependent L-type calcium channel (CACNA1S). As more than 300 variants of unknown significance have been detected to date, we examined whether freely available pathogenicity prediction tools are able to detect relevant MH causing variants. In this diagnostic accuracy study, blood samples from 235 individuals with a history of a clinical malignant hyperthermia or their close relatives were genetically screened for RYR1 variants of all 106 RYR1 exons and additionally for known variants of CACNA1S. In vitro contracture tests were conducted on muscle biopsies obtained from all individuals, independently of whether a pathogenic variant, a variant of unknown significance or no variant was detected. Comparisons were made to three established bioinformatic pathogenicity detection tools to identify the clinical impact of the variants of unknown significance. All detected genetic variants were tested for pathogenicity by three in silico approaches and compared to the in vitro contracture test. Sensitivity and specificity of exon screening of all individuals listed in our MH database was analyzed. Exon screening identified 97 (41%) of the 235 individuals as carriers of pathogenic variants. Variants of unknown significance were detected in 21 individuals. Variants of unknown significance were subdivided into 19 malignant-hyperthermia-susceptible individuals and 2 non-malignant-hyperthermia-susceptible individuals. All pathogenic variants as well as the malignant-hyperthermia-suspectible variants were correctly identified by the bioinformatic prediction tools. Sensitivity of in silico approaches ranged between 0.71 and 0.98 (Polyphen 0.94 [CI 95% 0.75; 0.99]; Sift 0.98 [CI 95% 0.81; 0.99]; MutationTaster 0.92 [CI 95% 0.75; 0.99]). Specificity differed depending on the used tool (Polphen 0.98 [CI 95% 0.32; 0.99]; Sift 0.98 [CI 95% 0.32; 0.99]; MutationTaster 0.00 [CI 95% 0.00; 0.60]). All pathogenic variants and variants of unknown significance were scored as probably damaging in individuals, demonstrating a high sensitivity. Specificity was very low in one of the three tested programs. However, due to potential genotype-phenotype discordance, bioinformatic prediction tools are currently of limited value in diagnosing pathogenicity of MH-susceptible variants.
| S-EPMC7873245 | biostudies-literature
Project description:Sequence-based testing of disease-susceptibility genes has identified many variants of unknown significance (VUSs) whose pathogenicity is unknown at the time of their measurement. Female breast cancer cases aged 20-49 years at diagnosis and who have VUSs in BRCA1 and no mutations in BRCA2 have previously been identified through the population-based Los Angeles County Cancer Surveillance Program. These nominal BRCA1 VUSs have been classified as "low," "medium," and "high" risk by four classification methods: Align-GVGD, Polyphen, Grantham matrix scores, and sequence conservation in mammalian species. Average hazard ratios (HRs) for classes of variants, i.e., the age-specific incidences of cancer for carriers of such variants divided by the population incidences, were estimated from the cancer family histories of first- and second-degree relatives of the index cases using modified segregation analysis. The study sample comprised 270 index cases and 4,543 of their relatives. There was weak evidence that the risk of breast cancer increases with the degree of sequence conservation (P = 0.03) and that missense variants at highly conserved sites are associated with a 5.6-fold (95 % confidence interval 1.4-22.2; P = 0.05) increased incidence of breast cancer. An upper bound of 2.3 is given for the average breast cancer HRs corresponding to variants classified as "low risk" by any of the four VUS classification methods. In summary, we have given a method to estimate cancer risks for groups of VUSs by combining existing classification methods with traditional penetrance analyses. This analysis suggests that classification methods for BRCA1 variants based on sequence conservation might be useful in a clinical setting. We have shown in principle that our method can be used to classify VUSs into clinically useful risk categories, but our specific findings should not be put into clinical practice unless confirmed by larger studies.
| S-EPMC4059774 | biostudies-literature
Project description:As the number of genes available for commercial sequencing increases and the promise of clinical whole-genome sequencing becomes a reality, the interpretation of the results of these tests becomes more challenging for the practicing neurologist as these studies have the potential to detect novel genetic variants. Such reports are becoming more frequent in general practice, and neurologists are often left to puzzle over the relevance of these "variants of unknown significance," as such genetic changes are often described, and how to communicate this information to the patients and their families. This article will briefly illustrate how clinicians can use such results in the care of their patients. Only genetic variants involving coding sequence will be considered, although similar methods may also be applied to changes such as noncoding alterations or copy number variations. It is also important to note that in some cases, particularly those involving tests that only sequence select exons, negative test results may also require special interpretation.
| S-EPMC3587691 | biostudies-literature
Project description:Contraction of striated muscles is driven by cyclic interactions of myosin head projecting from the thick filament with actin filament and is regulated by Ca2+ released from sarcoplasmic reticulum. Muscle thin filament consists of actin, tropomyosin and troponin, and Ca2+ binding to troponin triggers conformational changes of troponin and tropomyosin to allow actin-myosin interactions. However, the structural changes involved in this regulatory mechanism remain unknown. Here we report the structures of human cardiac muscle thin filament in the absence and presence of Ca2+ by electron cryomicroscopy. Molecular models in the two states built based on available crystal structures reveal the structures of a C-terminal region of troponin I and an N-terminal region of troponin T in complex with the head-to-tail junction of tropomyosin together with the troponin core on actin filament. Structural changes of the thin filament upon Ca2+ binding now reveal the mechanism of Ca2+ regulation of muscle contraction.
| S-EPMC6952405 | biostudies-literature
Project description: Not available
| EMPIAR-10348 | biostudies-other
Project description:BackgroundBRCA1 is a tumour suppressor with pleiotropic actions. Germline mutations in BRCA1 are responsible for a large proportion of breast-ovarian cancer families. Several missense variants have been identified throughout the gene but because of lack of information about their impact on the function of BRCA1, predictive testing is not always informative. Classification of missense variants into deleterious/high risk or neutral/low clinical significance is essential to identify individuals at risk.ObjectiveTo investigate a panel of missense variants.Methods and resultsThe panel was investigated in a comprehensive framework that included (1) a functional assay based on transcription activation; (2) segregation analysis and a method of using incomplete pedigree data to calculate the odds of causality; (3) a method based on interspecific sequence variation. It was shown that the transcriptional activation assay could be used as a test to characterise mutations in the carboxy-terminus region of BRCA1 encompassing residues 1396-1863. Thirteen missense variants (H1402Y, L1407P, H1421Y, S1512I, M1628T, M1628V, T1685I, G1706A, T1720A, A1752P, G1788V, V1809F, and W1837R) were specifically investigated.ConclusionsWhile individual classification schemes for BRCA1 alleles still present limitations, a combination of several methods provides a more powerful way of identifying variants that are causally linked to a high risk of breast and ovarian cancer. The framework presented here brings these variants nearer to clinical applicability.
| S-EPMC1735988 | biostudies-literature
Project description:Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation (TNNI1 R37C+/-) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in TNNI1 may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the TNNI1 R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca2+-binding sensitivity due to an increased Ca2+ off-rate constant. Furthermore, we generated TNNI1 R37C+/- mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca2+ transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in TNNI1 R37C+/- at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.
| S-EPMC6452669 | biostudies-literature
Project description:The cardiac thin filament regulates actomyosin interactions through calcium-dependent alterations in the dynamics of cardiac troponin and tropomyosin. Over the past several decades, many details of the structure and function of the cardiac thin filament and its components have been elucidated. We propose a dynamic, complete model of the thin filament that encompasses known structures of cardiac troponin, tropomyosin, and actin and show that it is able to capture key experimental findings. By performing molecular dynamics simulations under two conditions, one with calcium bound and the other without calcium bound to site II of cardiac troponin C (cTnC), we found that subtle changes in structure and protein contacts within cardiac troponin resulted in sweeping changes throughout the complex that alter tropomyosin (Tm) dynamics and cardiac troponin--actin interactions. Significant calcium-dependent changes in dynamics occur throughout the cardiac troponin complex, resulting from the combination of the following: structural changes in the N-lobe of cTnC at and adjacent to sites I and II and the link between them; secondary structural changes of the cardiac troponin I (cTnI) switch peptide, of the mobile domain, and in the vicinity of residue 25 of the N-terminus; secondary structural changes in the cardiac troponin T (cTnT) linker and Tm-binding regions; and small changes in cTnC-cTnI and cTnT-Tm contacts. As a result of these changes, we observe large changes in the dynamics of the following regions: the N-lobe of cTnC, the mobile domain of cTnI, the I-T arm, the cTnT linker, and overlapping Tm. Our model demonstrates a comprehensive mechanism for calcium activation of the cardiac thin filament consistent with previous, independent experimental findings. This model provides a valuable tool for research into the normal physiology of cardiac myofilaments and a template for studying cardiac thin filament mutations that cause human cardiomyopathies.
| S-EPMC3165030 | biostudies-literature
Project description:BACKGROUND:An emerging standard-of-care for long-QT syndrome uses clinical genetic testing to identify genetic variants of the KCNQ1 potassium channel. However, interpreting results from genetic testing is confounded by the presence of variants of unknown significance for which there is inadequate evidence of pathogenicity. METHODS AND RESULTS:In this study, we curated from the literature a high-quality set of 107 functionally characterized KCNQ1 variants. Based on this data set, we completed a detailed quantitative analysis on the sequence conservation patterns of subdomains of KCNQ1 and the distribution of pathogenic variants therein. We found that conserved subdomains generally are critical for channel function and are enriched with dysfunctional variants. Using this experimentally validated data set, we trained a neural network, designated Q1VarPred, specifically for predicting the functional impact of KCNQ1 variants of unknown significance. The estimated predictive performance of Q1VarPred in terms of Matthew's correlation coefficient and area under the receiver operating characteristic curve were 0.581 and 0.884, respectively, superior to the performance of 8 previous methods tested in parallel. Q1VarPred is publicly available as a web server at http://meilerlab.org/q1varpred. CONCLUSIONS:Although a plethora of tools are available for making pathogenicity predictions over a genome-wide scale, previous tools fail to perform in a robust manner when applied to KCNQ1. The contrasting and favorable results for Q1VarPred suggest a promising approach, where a machine-learning algorithm is tailored to a specific protein target and trained with a functionally validated data set to calibrate informatics tools.
| S-EPMC5679743 | biostudies-literature
Project description:Germline mutations in BRCA1/2 genes are responsible for a large proportion of hereditary breast and/or ovarian cancers. Many highly penetrant predisposition alleles have been identified and include frameshift or nonsense mutations that lead to the translation of a truncated protein. Other alleles contain missense mutations, which result in amino acid substitution and intronic variants with splicing effect. The discovery of variants of uncertain/unclassified significance (VUS) is a result that can complicate rather than improve the risk assessment process. VUSs are mainly missense mutations, but also include a number of intronic variants and in-frame deletions and insertions. Over 2,000 unique BRCA1 and BRCA2 missense variants have been identified, located throughout the whole gene (Breast Cancer Information Core Database (BIC database)). Up to 10-20% of the BRCA tests report the identification of a variant of uncertain significance. There are many methods to discriminate deleterious/high-risk from neutral/low-risk unclassified variants (i.e., analysis of the cosegregation in families of the VUS, measure of the influence of the VUSs on the wild-type protein activity, comparison of sequence conservation across multiple species), but only an integrated analysis of these methods can contribute to a real interpretation of the functional and clinical role of the discussed variants. The aim of our manuscript is to review the studies on BRCA VUS in order to clarify their clinical relevance.
| S-EPMC3837329 | biostudies-literature