A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: application of the CD3/CD28 assay in lymphocytes.
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.
Tada
Hayato
H
Division of Cardiovascular Medicine, Department of Internal Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan. ht240z@med.kanazawa-u.ac.jp
Kawashiri
Masa-aki
MA
Noguchi
Tohru
T
Mori
Mika
M
Tsuchida
Masayuki
M
Takata
Mutsuko
M
Nohara
Atsushi
A
Inazu
Akihiro
A
Kobayashi
Junji
J
Yachie
Akihiro
A
Mabuchi
Hiroshi
H
Yamagishi
Masakazu
M
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
D000906:Antibodies; D018106:CD28 Antigens; D017252:CD3 Complex; D008074:Lipoproteins; D011973:Receptors, LDL; D006493:Heparin