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Results for “Functional assay”

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At least 19 recordsLinked to original sources
PubMed2025

A functional assay to classify RB1 variants of uncertain significance.

PURPOSE: The RB1 gene encodes the retinoblastoma protein (pRB) playing a major role in cell cycle control, particularly by its interaction with E2F transcription factors. Familial forms of retinoblastoma are caused by germline pathogenic variants in the RB1 gene predisposing to retinoblastoma and other tumors. By analyzing the RB1 gene in patients with retinoblastoma, we found that missense variants often remain variants of uncertain significance (VUS). METHODS: To classify RB1 VUS, we developed a functional assay evaluating their impact on the ability of pRB to inhibit the activity of the E2F1 promoter, with a luciferase reporter gene. A set of 14 pathogenic/likely pathogenic and benign/likely benign RB1 variants was used for validation. RESULTS: We tested 16 VUS detected in patients with retinoblastoma and found that 9 VUS reduced the ability of pRB to inhibit E2F1 promoter. Among them, the (RB1) c.2263T>G p.(Phe755Val) variant showed a reduced level of pRB on Western blot, suggesting a defect in pRB stability. By applying the criterion PS3_moderate of the American College of Medical Genetics and Genomics/Association for Molecular Pathology classification to this functional assay, 5 of the 9 VUS with functional impact could be classified as likely pathogenic. CONCLUSION: This functional assay can improve the molecular diagnosis of retinoblastoma predisposition by a better determination of pathogenic/likely pathogenic RB1 variants.

Humans↗
PubMed2026

'Truthsets' for clinical validation of large-scale functional assays: Practice recommendations from Cancer Variant Interpretation Group UK (CanVIG-UK).

BACKGROUND: Large-scale functional assays, including multiplex assays of variant effect, have substantial potential to resolve variants of uncertain significance (VUS), particularly for rare missense variants where clinical and population evidence are limited. The ClinGen assay-level clinical validation framework described by Brnich et al provided baseline guidance for the use of functional data for variant classification. However, clear consensus regarding construction of variant 'truthsets' by which to clinically validate functional data remains lacking. METHODS: CanVIG-UK developed consensus recommendations for truthset construction through an iterative national consultation process involving the CanVIG Steering Advisory Group (CStAG), wider CanVIG-UK membership, and engagement with international functional genomics experts. Consultation was based on previous analyses of 2,120 truthset constructions examining the impact of truthset composition on evidence point allocation within the ClinGen assay-level clinical validation framework. RESULTS: Across several consultations, CanVIG-UK established nine guiding principles and seven best-practice recommendations for assay-level clinical validation, using the assumed context of an assay for a cancer susceptibility gene where loss-of-function is the mechanism of pathogenicity. The principal recommendation stipulates, where assays are intended for use in interpretation of largely missense variants, the truthset used to validate should comprise only missense variants. Rather than mixtures of different variant types which may serve to over-estimate assay performance. Additional recommendations support option for relaxation of truthset stringency to improve power, augmentation of benign missense truthsets with systematically derived 'proxy-clinical' benign variants, independent clinical validation separate from assayist-defined validation, and careful evaluation of missense score distributions against that of protein-truncating and synonymous variants. Guidance is also provided for scenarios with limited pathogenic truthset availability and for assays reporting multiple deleterious zones or readouts. CONCLUSIONS: The CanVIG-UK principles and recommendations for truthset construction upon the ClinGen assay-level clinical validation framework, while aiming to form a baseline for future discussion regarding other functional and disease contexts and helping to address the gap between publication of new data and routine clinical implementation.

Journal Article↗
PubMed2025

CryoSCAPE: Scalable immune profiling using cryopreserved whole blood for multi-omic single cell and functional assays.

BACKGROUND: The field of single cell technologies has rapidly advanced our comprehension of the human immune system, offering unprecedented insights into cellular heterogeneity and immune function. While cryopreserved peripheral blood mononuclear cell (PBMC) samples enable deep characterization of immune cells, challenges in clinical isolation and preservation limit their application in underserved communities with limited access to research facilities. We present CryoSCAPE (Cryopreservation for Scalable Cellular And Proteomic Exploration), a scalable method for immune studies of human PBMC with multi-omic single cell assays using direct cryopreservation of whole blood. RESULTS: Comparative analyses of matched human PBMC from cryopreserved whole blood and density gradient isolation demonstrate the efficacy of this methodology in capturing cell proportions and molecular features. The method was then optimized and verified for high sample throughput using fixed single cell RNA sequencing and liquid handling automation with a single batch of 60 cryopreserved whole blood samples. Additionally, cryopreserved whole blood was demonstrated to be compatible with functional assays, enabling this sample preservation method for clinical research. CONCLUSIONS: The CryoSCAPE method, optimized for scalability and cost-effectiveness, allows for high-throughput single cell RNA sequencing and functional assays while minimizing sample handling challenges. Utilization of this method in the clinic has the potential to democratize access to single-cell assays and enhance our understanding of immune function across diverse populations.

Humans↗
PubMed2025

Using the Tether Function Assay to Identify Potential Regulators of mRNA Translation and mRNA Decay.

RNA binding proteins (RBPs) and their associated partners are key factors of posttranscriptional control of gene expression. To study and manipulate the functional consequences of binding of these regulators to their targets, several tethering assays have been developed, in which a protein of interest is brought to a reporter mRNA through heterologous RNA-protein interaction motifs. The effect of such constrained binding is then monitored by measuring the accumulation of the reporter protein and mRNA. This chapter describes a protocol for the λN-BoxB tether system in transiently transfected mammalian cells. Combining the luciferase reporter technology to quantify protein amounts by light measurement and RNA amounts by RT-qPCR, this assay provides a simple and robust way to analyze the consequences of any protein binding in a controlled and defined manner.

RNA, Messenger↗
PubMed1978

A radial hemolysis method in agarose for the functional assay of properdin.

A simple one-step radial hemolytic assay for properdin has been devised. In this assay, the test material is introduced into a well in an agarose plate containing optimal concentrations of normal human serum immunochemically depleted of properdin (RP), EGTA, magnesium ions and unsensitized guinea pig erythrocytes. Following radial diffusion, the area of the hemolytic zones resulting from the activation of the alternative complement pathway and bystander lysis of guinea pig erythrocytes was directly proportional to the concentration of properdin in the test material. The assay is specific reproducible and sensitive and the correlation with the radioimmunoassay for properdin is very good. The assay has been used to measure properdin activity in animal sera.

Animals↗
PubMed1977

Functional assay of cytotoxic lymphocytes involved in antibody-mediated cytotoxicity in normal and rheumatoid subjects.

Antibody-mediated lymphocyte-induced cytotoxicity (K cell activity) was measured in a system consisting of Chang human liver cells, rabbit anti-Chang antiserum, and Triosil-Ficoll purified human lymphocytes. It was concluded that one K cell probably inactivates or kills one target cell, and that the number of active K cells is similar in the peripheral blood of normal and rheumatoid subjects and is about 0-5%.

Antibodies↗
PubMed2026

Automated patch clamp data improve variant classification and penetrance stratification for SCN5A-Brugada syndrome.

BACKGROUND AND AIMS: Brugada Syndrome (BrS) is an inherited arrhythmia disorder that causes an elevated risk of sudden cardiac death. Approximately 20% of patients with BrS have rare variants in SCN5A, which encodes the cardiac sodium channel NaV1.5. Genetic workup of BrS is often complicated by SCN5A variants of uncertain significance (VUS) and/or incomplete penetrance. This study deployed an SCN5A-BrS functional assay at cohort scale to facilitate the implementation of genetic and precision medicine. METHODS: All 252 missense and in-frame insertion/deletion SCN5A variants from a previously published large cohort of BrS cases (n = 3335 patients) were analysed using a calibrated high-throughput automated patch-clamp (APC) assay. Variant functional Z-scores were assigned evidence levels ranging from BS3_moderate (normal function) to PS3_strong (loss-of-function), as defined by American College of Medical Genetics and Genomics criteria. Functional evidence was combined with population frequency, hotspot, case counts, protein-length changes, and in silico predictions. Odds ratios of BrS case-control enrichment and penetrance for BrS were calculated from variant frequencies in the BrS cohort and in gnomAD. RESULTS: Most variants (146/252) were functionally abnormal (Z ≤ -2), with 100 having severe loss-of-function (Z ≤ -4). Functional evidence enabled the reclassification of 110 of 225 VUS; 104 to likely pathogenic and 6 to likely benign. SCN5A variants with loss-of-function were mainly localized to the transmembrane domains, especially the regions comprising the central pore. SCN5A variant penetrance was proportional to the severity of loss-of-function; variants with Z ≤ -6 had penetrance of 24.5% (15.9%-37.7% CI) and an odds ratio of 501 for BrS. CONCLUSIONS: This cohort-scale APC dataset stratifies SCN5A variants found in BrS patients into normal function 'bystander' variants that have a low risk of BrS and loss-of-function variants that have a high risk for BrS. Functional data can be integrated with other criteria to reclassify a substantial fraction of VUS. The dataset helps clarify the SCN5A-BrS relationship and will improve the diagnosis and clinical management of BrS probands and their families.

Humans↗
PubMed1978

Desorption assay: a functional in vitro test for measuring the adhesion of E. coli on the urinary tract epithelium.

A functional test was developed to measure different adhesive properties of various Escherichia coli strains on the urinary tract epithelium. The test is based on initial incubation of everted canine ureteral segments in 2-hr cultures of E. coli (adsorption period), followed by sequential elutions in phosphate buffered saline. The release of bacteria (desorption rate) was measured from each elution by quantitative bacteriology. The function of the desorption assay was tested by showing different adhesive properties of various strains of E. coli. The strains isolated from infected canine urinary tracts showed high adhesion capacities superior to other strains of E. coli from different animal and/or tissue origin. This indicates a selective tissue and animal trophism determined by the strength of mucosal attachment. The test is also a valuable tool for measuring adhesion inhibiting substances in urine. The bulk of the adhesion inhibiting effect was localized in the urine ultrafiltrate.

Adhesiveness↗
PubMed1979

Monocytes and macrophages in malignant melanoma IV. Effects of C. parvum on monocyte function.

Assays for the capacity of peripheral-blood monocytes (a) to mature in vitro into macrophages, (b) to reduce nitro-blue tetrazolium (NBT) and (c) to lyse antibody-coated human Group A red cells, were applied to a group of 82 patients with histologically proven malignant melanoma. In patients with micrometastatic disease there was an enhancement of red-cell lysis and NBT reduction, suggesting that their monocytes are in some way "activated", whereas NBT reduction was suppressed in those with overt dissemination. Monocyte maturation in vitro was impaired in all patient groups to an extent which correlated with overall tumour burden. Corynebacterium parvum was administered i.v. to 12 patients with disseminated disease and by the intradermal route to 24 patients with micrometastatic disease. The 3 monocyte functions were significantly enhanced by C. parvum.

Bacterial Vaccines↗
PubMed1975

Hereditary C6 deficiency in man.

An 18-year-old black female (D.B.) in good general health was found to have no hemolytic activity in serum CH50 titrations. Functional assays yielded normal values for all C components except C6. C6 was not detectable in plasma or serum by two different functional assays nor by antigenic analysis using monospecific anti-C6 antibody. Hemolytic activity was restored by addition of functionally pure C6. By specific functional assay, both parents and 5 of 6 available sibs had approximately half-normal serum C6 levels and 1 sib was normal. Biologic properties of D.B. serum include: a) absent bactericidal activity against S. typhi 0 901 with or without added rabbit antibody; b) normal generation of chemotactic activity for human neutrophils in the presence of endotoxin or aggregated IgG; c) ability to sensitize appropriate cells for immune adherence of agglutination by anti-C3 Coombs serum; and d) inability to lyse PNH red cells in either acid hemolysis or "sugar water" tests. An extensive clotting workup by standard methods was within normal limits. These studies document for the first time a human kindred with C6 deficiency. This defect exhibits a classic mendelian autosomal inheritance, with all 3 genotypes being recognizable. Unlike the C6-deficient rabbits studied by others, the homozygous C6-deficient human exhibits chemotactic and coagulation functions within the range of normal.

Adolescent↗
PubMed2025

A functional enzymatic assay as potential readout for a clinical trial of a schistosomiasis vaccine.

An estimated 200 million people are currently infected with schistosomiasis and an additional 800 million reside in high transmission-risk areas in 78 endemic countries. In this report we describe a functional enzymatic assay based on the core calpain antigen (Sm-p80) of the schistosomiasis vaccine, SchistoShield®. A 44 kDa soluble variant of the core Sm-p80 antigen (B7), was assessed for its enzymatic activity using a fluorescent synthetic substrate. Inhibition of the B7 enzymatic activity by Sm-p80-specific antibodies obtained from pre-clinical trials in rodents, non-human primates as well as from participants of the human clinical trials was measured. The B7 enzyme activity followed a Michaelis-Menten-like kinetic behavior. Statistically significant inhibition of the B7 activity was observed by Sm-p80-specific antibodies produced by immunized mice, non-human primates and humans. This quantitative serological assay could be of value in assessing the effectiveness of the SchistoShield® vaccine in human trials in Africa.

Journal Article↗