title: An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations.
abstract: The main causes of familial hypercholesterolemia (FH) are mutations in LDL receptor (LDLR) gene. Functional studies are necessary to demonstrate the LDLR function impairment caused by mutations and would be useful as a diagnostic tool if they allow discrimination between FH patients and controls. In order to identify the best method to detect LDLR activity, we compared continuous Epstein-Barr virus (EBV)-transformed B-lymphocytes and mitogen stimulated T-lymphocytes. In addition, we characterized both novel and known mutations in the LDLR gene. T-lymphocytes and EBV-transformed B-lymphocytes were obtained from peripheral blood of 24 FH patients and 24 control subjects. Functional assays were performed by incubation with fluorescent LDL followed by flow cytometry analysis. Residual LDLR activity was calculated normalizing fluorescence for the mean fluorescence of controls. With stimulated T-lymphocytes we obtained a better discrimination capacity between controls and FH patients compared with EBV-transformed B-lymphocytes as demonstrated by receiver operating characteristic (ROC) curve analysis (the areas under the curve are 1.000 and 0.984 respectively; P < 0.0001 both). The characterization of LDLR activity through T-lymphocytes is more simple and faster than the use of EBV-transformed B-lymphocytes and allows a complete discrimination between controls and FH patients. Therefore the evaluation of residual LDLR activity could be helpful not only for mutation characterization but also for diagnostic purposes.
authors.affiliation: CEINGE - Biotecnologie Avanzate, Napoli, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Romano
authors.forename: Maria Donata
authors.initials: MD
authors.lastname: Di Taranto
authors.forename: Peppino
authors.initials: P
authors.lastname: Mirabelli
authors.forename: Maria Nicoletta
authors.initials: MN
authors.lastname: D'Agostino
authors.forename: Arcangelo
authors.initials: A
authors.lastname: Iannuzzi
authors.forename: Gennaro
authors.initials: G
authors.lastname: Marotta
authors.forename: Marco
authors.initials: M
authors.lastname: Gentile
authors.forename: Maddalena
authors.initials: M
authors.lastname: Raia
authors.forename: Rosa
authors.initials: R
authors.lastname: Di Noto
authors.forename: Luigi
authors.initials: L
authors.lastname: Del Vecchio
authors.forename: Paolo
authors.initials: P
authors.lastname: Rubba
authors.forename: Giuliana
authors.initials: G
authors.lastname: Fortunato
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics; D008077:Lipoproteins, LDL / Q000378:metabolism; D008934:Mitogens / Q000494:pharmacology*; D012372:ROC Curve; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; D008934:Mitogens; D011973:Receptors, LDL