Comparison of the Genetic Defect with LDL-Receptor Activity in Cultured Cells from Patients With a Clinical Diagnosis of Heterozygous Familial Hypercholesterolemia

Sun, Xi‐Ming; Patel, Dilip D.; Knight, Brian L.; Soutar, Anne K.

doi:10.1161/01.atv.17.11.3092

Cited by 52 publications

(30 citation statements)

References 33 publications

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“…To our knowledge, only few studies have investigated the relationship between LDLR genotype and its activity (20). Although there have been a number of studies on LDLR activity measurements in FH using DiI-LDL uptake in blood peripheral lymphocytes, some causes of defective LDLR activity, such as the K790X mutation, often appear to have been overlooked.…”

Section: Discussionmentioning

confidence: 99%

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

Tada

Kawashiri

Noguchi

et al. 2009

Clinica Chimica Acta

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Abbreviated title: An improved assay for LDL receptor activity using anti-CD3/CD28 beads Abbreviations: DiI, 3,3"--dioctadecylindocarbocyanin; FH, familial hypercholesterolemia; NARC-1, neural apoptosis-regulated convertase 1; FITC, fluorescein isothiocyanate; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; LPDS, lipoprotein deficient serum; rIL-2, recombinant interleukin-2; MF, mean fluorescence; TC, total cholesterol; CV, coefficient of variation. 3 AbstractBackground: 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 two 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 differenciated 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.Conclusion: These results demonstrate that our new method is applicable for measuring LDLR 4 activity, even in subjects with an internally defective allele, and that T-lymphocytes of the FH K790X mutation possess characteristics of that allele.

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Section: Discussionmentioning

confidence: 99%

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

Tada

Kawashiri

Noguchi

et al. 2009

Clinica Chimica Acta

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“…Other detection systems such as Denaturing Gradient Gel Electrophoresis (DGGE) have been used in mutation screening of the LDLR 43 ± 45 without achieving detection of mutations in all patients. The detection rate in one study using DGGE was 81% 45 although the sample size was small (n=32) and the clinical criteria were very strict, for example total cholesterol 49.5 mmol/l. This confirms the importance of the clinical diagnosis with respect to the detection rate.…”

Section: Mutation Detection Ratesmentioning

confidence: 98%

A molecular genetic service for diagnosing individuals with familial hypercholesterolaemia (FH) in the United Kingdom

et al. 2001

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A genetic diagnostic service for familial hypercholesterolaemia (FH) has been established over the last 4 years in the Clinical Molecular Genetics Laboratory at Great Ormond Street Hospital for Children NHS Trust (GOSH), London. In total there have been 368 referrals; 227 probands and 141 family members, which have come from a number of lipid clinics and from general practitioners. FH is caused by mutations in the lowdensity lipoprotein receptor gene (LDLR) and these are analysed by SSCP, DNA sequencing and direct assays. The clinically indistinguishable disorder, familial defective apolipoprotein B100 (FDB) is caused by one of three mutations in the apolipoprotein B100 gene (APOB) which are analysed by direct assays. Mutations predicted to be pathogenic were found in 76 probands, 67 in LDLR (23 previously undescribed) and nine in APOB. The mutation detection rate was 53% in paediatric probands, 32% in adults with a`definite' FH diagnosis (tendon xanthoma positive) and 14% in adults with a`possible' FH diagnosis (tendon xanthoma negative). The predicted loss of sensitivity that would result from reducing the number of exons tested has been assessed, and a molecular screening strategy suitable for UK patients is proposed. A similar strategy may be useful for other countries where genetic heterogeneity results in a wide mutation spectrum for FH.

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“…This discovery is not surprising, as other studies have reported that a certain proportion of putative FH subjects do not have detectable mutations at the LDL-R locus. [33][34][35] From the mutation list shown in Table II, the conclusion that emerges is that Italy is like the United Kingdom, 7,33 Germany, 36 and Japan, 37 where many mutations have produced a highly heterogeneous picture, in contrast to the situation found in other countries (like Norway, Finland, and, to some extent, Denmark), where few mutations account for a large proportion of FH patients. 38 -40 For each index case, we tried to retrieve as much family data as possible to define the geographic origin of the ancestor likely to carry the mutation identified in the index case.…”

Section: Mutation Spectrum and Clustersmentioning

confidence: 99%

Clinical Expression of Familial Hypercholesterolemia in Clusters of Mutations of the LDL Receptor Gene That Cause a Receptor-Defective or Receptor-Negative Phenotype

Bertolini¹,

Cantàfora²,

Averna³

et al. 2000

ATVB

133

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Abstract-Seventy-one mutations of the low density lipoprotein (LDL) receptor gene were identified in 282 unrelated Italian familial hypercholesterolemia (FH) heterozygotes. By extending genotype analysis to families of the index cases, we identified 12 mutation clusters and localized them in specific areas of Italy. To evaluate the impact of these mutations on the clinical expression of FH, the clusters were separated into 2 groups: receptor-defective and receptor-negative, according to the LDL receptor defect caused by each mutation. These 2 groups were comparable in terms of the patients' age, sex distribution, body mass index, arterial hypertension, and smoking status. In receptor-negative subjects, LDL cholesterol was higher (ϩ18%) and high density lipoprotein cholesterol lower (Ϫ5%) than the values found in receptor-defective subjects. The prevalence of tendon xanthomas and coronary artery disease (CAD) was 2-fold higher in receptor-negative subjects. In patients Ͼ30 years of age in both groups, the presence of CAD was related to age, arterial hypertension, previous smoking, and LDL cholesterol level. Independent contributors to CAD in the receptor-defective subjects were male sex, arterial hypertension, and LDL cholesterol level; in the receptor-negative subjects, the first 2 variables were strong predictors of CAD, whereas the LDL cholesterol level had a lower impact than in receptor-defective subjects. Overall, in receptor-negative subjects, the risk of CAD was 2.6-fold that of receptordefective subjects. Wide interindividual variability in LDL cholesterol levels was found in each cluster. Apolipoprotein E genotype analysis showed a lowering effect of the ⑀2 allele and a raising effect of the ⑀4 allele on the LDL cholesterol level in both groups; however, the apolipoprotein E genotype accounted for only 4% of the variation in LDL cholesterol.Haplotype analysis showed that all families of the major clusters shared the same intragenic haplotype cosegregating with the mutation, thus suggesting the presence of common ancestors. (Arterioscler Thromb Vasc Biol. 2000;20:e41-e52.)

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Comparison of the Genetic Defect with LDL-Receptor Activity in Cultured Cells from Patients With a Clinical Diagnosis of Heterozygous Familial Hypercholesterolemia

Cited by 52 publications

References 33 publications

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

A molecular genetic service for diagnosing individuals with familial hypercholesterolaemia (FH) in the United Kingdom

Clinical Expression of Familial Hypercholesterolemia in Clusters of Mutations of the LDL Receptor Gene That Cause a Receptor-Defective or Receptor-Negative Phenotype

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