title: A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: application of the CD3/CD28 assay in lymphocytes.
abstract: BACKGROUND
  The objective of this study was to develop a new and simple method for measuring low-density lipoprotein receptor (LDLR) activity using peripheral lymphocytes enabling us to clinically diagnose familial hypercholesterolemia (FH) and ascertain the involved mutations (such as K790X mutation), that might not be clearly detected in the conventional method.
  
  
  METHODS
  Our method comprised the following 2 features: first, we used anti-CD3/CD28 beads to stimulate T-lymphocytes to obtain a uniform fraction of lymphocytes and maximum up-regulation of LDLR. Second, we excluded the possibility of overestimation of lymphocyte signals bound only to its surface, by adding heparin to the cultured lymphocytes used for the LDLR assay.
  
  
  RESULTS
  Based on the genetic mutation, the FH subjects were divided into 2 groups, K790X, (n=20) and P664L, (n=5), and their LDLR activities was measured by this method, which was found to be 55.3+/-8.9% and 63.9+/-13.8%, respectively, of that of the control group (n=15). In comparison, the LDLR activity was 86.1+/-11.6% (K790X) and 73.3+/-6.3% (P664L) of that of the control group when measured by the conventional method, indicating that impairment of LDLR function in FH K790X subjects was much more clearly differentiated with our method than with the conventional method (paired t-test, p<0.0001). The levels of LDLR expression also showed similar tendencies, that is, 89.4+/-13.2% (K790X) and 76.9+/-17.4% (P664L) of that of the control group when measured by the conventional method, and 78.1+/-9.7% (K790X) and 70.3+/-26.5% (P664L) when measured by our new method. In addition, we confirmed that there was little influence of statin treatment on LDLR activity among the study subjects when our method was used.
  
  
  CONCLUSIONS
  These results demonstrate that our new method is applicable for measuring LDLR activity, even in subjects with an internally defective allele, and that T-lymphocytes of the FH K790X mutation possess characteristics of that allele.
authors.affiliation: Division of Cardiovascular Medicine, Department of Internal Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan. ht240z@med.kanazawa-u.ac.jp
authors.forename: Hayato
authors.initials: H
authors.lastname: Tada
authors.forename: Masa-aki
authors.initials: MA
authors.lastname: Kawashiri
authors.forename: Tohru
authors.initials: T
authors.lastname: Noguchi
authors.forename: Mika
authors.initials: M
authors.lastname: Mori
authors.forename: Masayuki
authors.initials: M
authors.lastname: Tsuchida
authors.forename: Mutsuko
authors.initials: M
authors.lastname: Takata
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nohara
authors.forename: Akihiro
authors.initials: A
authors.lastname: Inazu
authors.forename: Junji
authors.initials: J
authors.lastname: Kobayashi
authors.forename: Akihiro
authors.initials: A
authors.lastname: Yachie
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mabuchi
authors.forename: Masakazu
authors.initials: M
authors.lastname: Yamagishi
mesh_terms: D000483:Alleles; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001774:Blood Chemical Analysis / Q000379:methods*; D018106:CD28 Antigens / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D005260:Female; D006493:Heparin / Q000378:metabolism; D006579:Heterozygote; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000097:blood* / Q000175:diagnosis / Q000235:genetics / Q000473:pathology; D008074:Lipoproteins / Q000172:deficiency; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D011973:Receptors, LDL / Q000097:blood / Q000378:metabolism*; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D018106:CD28 Antigens; D017252:CD3 Complex; D008074:Lipoproteins; D011973:Receptors, LDL; D006493:Heparin