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.

An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations / Romano, M; DI TARANTO, MARIA DONATA; Mirabelli, P; D'Agostino, MARIA NICOLETTA; Iannuzzi, A; Marotta, Gennaro; Gentile, M; Raia, M; DI NOTO, Rosa; DEL VECCHIO, Luigi; Rubba, PAOLO OSVALDO FEDERICO; Fortunato, Giuliana. - In: JOURNAL OF LIPID RESEARCH. - ISSN 0022-2275. - 52:11(2011), pp. 2095-2100. [10.1194/jlr.D017772.]

An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations.

Di Taranto MD;D'AGOSTINO, MARIA NICOLETTA;MAROTTA, GENNARO;DI NOTO, ROSA;DEL VECCHIO, LUIGI;RUBBA, PAOLO OSVALDO FEDERICO;FORTUNATO, GIULIANA
2011

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.
2011
An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations / Romano, M; DI TARANTO, MARIA DONATA; Mirabelli, P; D'Agostino, MARIA NICOLETTA; Iannuzzi, A; Marotta, Gennaro; Gentile, M; Raia, M; DI NOTO, Rosa; DEL VECCHIO, Luigi; Rubba, PAOLO OSVALDO FEDERICO; Fortunato, Giuliana. - In: JOURNAL OF LIPID RESEARCH. - ISSN 0022-2275. - 52:11(2011), pp. 2095-2100. [10.1194/jlr.D017772.]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/576102
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