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T P Leren

Publications and source records attributed to T P Leren.

At least 19 recordsLinked to original sources
PubMed2006

Low-density lipoprotein receptor activity in Epstein-Barr virus-transformed lymphocytes from heterozygotes for the D374Y mutation in the PCSK9 gene.

OBJECTIVE: Missense mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have been found to cause autosomal dominant hypercholesterolemia. The objective of this study was to investigate possible mechanisms by which mutation D374Y in the PCSK9 gene causes hypercholesterolemia. MATERIAL AND METHODS: Binding and internalization of low-density lipoprotein LDL in Epstein-Barr virus (EBV)-transformed lymphocytes from D374Y heterozygotes were examined. The autocatalytic activity of the D374Y mutant was studied in transiently transfected HEK293 cells. RESULTS: As determined by Western blot analysis of transiently transfected HEK293 cells, the autocatalytic activity of the D374Y mutant was approximately 95% of the wild-type. Levels of PCSK9 mRNA in EBV-transformed lymphocytes from D374Y heterozygotes and normal controls were similar and less than 1/1000 of the level in HepG2 cells. The amount of cell surface LDL receptors (LDLRs) in EBV-transformed lymphocytes from five D374Y heterozygotes was non-significantly increased by 17% compared with the amount in normal controls. LDLR-dependent binding and internalization of LDL in EBV-transformed lymphocytes from D374Y heterozygotes were non-significantly reduced by 11% and 12%, respectively, compared to the corresponding values in normal controls. CONCLUSIONS: LDLR-mediated endocytosis of LDL is not reduced in EBV-transformed lymphocytes from D374Y heterozygotes. Because of the extremely low levels of PCSK9 mRNA in EBV-transformed lymphocytes, it is possible that the LDLR-dependent endocytosis of LDL could be more severely affected in hepatocytes from D374Y heterozygotes than in EBV-transformed lymphocytes.

Cell Line, Transformed↗
PubMed2004

Mutations in the PCSK9 gene in Norwegian subjects with autosomal dominant hypercholesterolemia.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is at a locus for autosomal dominant hypercholesterolemia, and recent data indicate that the PCSK9 gene is involved in cholesterol biosynthesis. Mutations within this gene have previously been found to segregate with hypercholesterolemia. In this study, DNA sequencing of the 12 exons of the PCSK9 gene has been performed in 51 Norwegian subjects with a clinical diagnosis of familial hypercholesterolemia where mutations in the low-density lipoprotein receptor gene and mutation R3500Q in the apolipoprotein B-100 gene had been excluded. Two novel missense mutations were detected in the catalytic subdomain of the PCSK9 gene. Two patients were heterozygotes for D374Y, and one patient was a double heterozygote for D374Y and N157K. D374Y segregated with hypercholesterolemia in the two former families where family members were available for study. Our findings support the notion that mutations in the PCSK9 gene cause autosomal dominant hypercholesterolemia.

Adolescent↗
PubMed2004

Cascade genetic screening for familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is caused by a mutation in the low-density lipoprotein receptor gene and is characterized by hypercholesterolemia, xanthomas, and premature coronary heart disease. Heterozygotes typically have values for total serum cholesterol in the range of 7-15 mmol/l and efficient lipid-lowering drug therapy is available. However, only approximately 20% of patients are diagnosed and less than 10% are being adequately treated. The most cost-effective strategy to diagnose patients with FH is to screen close relatives of patients already diagnosed with FH. This is referred to as cascade genetic screening. This review focuses on organization of a cascade genetic screening program for FH as well as cost-efficiency assessments, health benefits, possible adverse effects, and the screening of children. The author concludes that cascade genetic screening for FH leads to health benefits and is cost-effective without causing psychological or social damage. Accordingly, national cascade genetic screening programs for FH should be part of ordinary health care.

Cost-Benefit Analysis↗
PubMed2000

The genetic algorithm applied to haplotype data at the LDL receptor locus.

Conventional statistical methods based upon single restriction fragment length polymorphisms often prove inadequate in studies of genetic variation. Cladistic analysis has been suggested as an alternative, but requires basic assumptions that usually cannot be met. We wanted to test whether it could be a workable approach to apply the genetic algorithm, an artificial intelligence method, to haplotype data. The genetic algorithm creates in-computer artificial 'individuals', all having 'genes' coding for solutions to a problem. The individuals are allowed to compete and 'mate', individuals with genes coding for better solutions mating more often. Genes coding for good solutions survive through generations of the genetic algorithm. At the end of the run, the best solutions can be extracted. We applied the genetic algorithm to data consisting of cholesterol values and haplotypes made up of seven restriction sites at the LDL receptor locus. The persons included were 114 FH (familial hypercholesterolemia) patients and 61 normals. The genetic algorithm found the restriction sites 1 (Sph1 in intron 6), 2 (StuI in exon 8), and 7 (ApaLI site in the 3' flanking region) were associated with high cholesterol levels. As a validity check we used runs of the genetic algorithm applied to 'artificial patients', i.e. artificially generated haplotypes linked to artificially generated cholesterol values. This demonstrated the genetic algorithm consistently found the appropriate haplotype. We conclude that the genetic algorithm may be a useful tool for studying genetic variation.

Algorithms↗
PubMed2000

Does maternal or paternal heredity affect carotid atherosclerosis in children with familial hypercholesterolaemia?

Maternal hypercholesterolaemia has recently been shown to increase the number of fatty streaks in the foetal aorta, as well as subsequent progression of fatty streaks to more advanced lesions. We compared carotid intima-media thickness by B-mode ultrasonography in children who inherited familial hypercholesterolaemia from their mothers or fathers, hypothesizing that children whose mothers were hypercholesterolaemic during pregnancy had increased atherogenesis compared with children whose mothers did not have familial hypercholesterolaemia. We found that carotid intima-media thickness and prevalence of plaque did not differ between children with familial hypercholesterolaemia who inherited FH maternally or paternally.

Adolescent↗
PubMed2000

A functional polymorphism in the promoter region of the microsomal triglyceride transfer protein (MTP -493G/T) influences lipoprotein phenotype in familial hypercholesterolemia.

The microsomal triglyceride transfer protein (MTP) has a key function in intracellular apolipoprotein (apo) B lipidation and secretion of very low density lipoprotein (VLDL). A recently discovered functional polymorphism in the promoter of the MTP gene (-493G/T) affects the plasma concentration of low density lipoprotein (LDL) cholesterol and the VLDL distribution between large and small particle species in healthy men. This phenotype is likely to be explained by an effect on VLDL synthesis. Against this background, we studied the effect of the MTP-493G/T polymorphism in a large cohort (217 men and 211 women) with heterozygous familial hypercholesterolemia (FH). A 40% to 50% lower serum triglyceride level was observed in homozygous carriers of the MTP-493 T allele (T/T, 0.93+/-0.34; G/T, 1.54+/-1.40; and G/G, 1.56+/-1.24 mmol/L; T/T vs G/T P=0.04, T/T vs G/G P=0.02). In contrast to the situation in healthy subjects, the MTP promoter polymorphism did not have a significant effect on the LDL cholesterol levels in FH subjects, although the same trend was observed (T/T, 7.31+/-1.87; G/T, 7. 80+/-2.12; and G/G, 7.91+/-2.31 mmol/L, NS). Adjustment for the apo E gene polymorphism by inclusion of subjects homozygous for the apo E3 allele only revealed a reciprocal high density lipoprotein cholesterol-elevating effect (T/T, 1.41+/-0.73; G/T, 1.18+/-0.27; and G/G, 1.16+/-0.29 mmol/L; T/T vs G/T P=0.06, T/T vs G/G P=0.04). This effect seemed to be sex-specific because it was accounted for by the female patients. In conclusion, the LDL cholesterol-lowering effect of the rare MTP gene promoter variant (MTP-493T) present in healthy subjects is shifted to a triglyceride-lowering effect in FH. These data suggest that the MTP gene has a role in modulating the clinical phenotype of FH.

Adolescent↗
PubMed1999

Mutant transcripts of the LDL receptor gene: mRNA structure and quantity.

mRNA of the low-density lipoprotein receptor (LDLR) gene from 22 heterozygous familial hypercholesterolemic subjects possessing different mutations in this gene was analyzed by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) in order to detect abnormally spliced transcripts. These analyses revealed abnormally spliced transcripts for the two splice-site mutations 1359-1G-->A and 1705 + 1G-->T. The abnormally spliced transcript for mutation 1359-1G-->A was caused by activation of a cryptic acceptor splice site in exon 10. As a result, seven nucleotides of exon 10 were deleted. For mutation 1705 + 1G-->T, two mutant transcripts were observed. In the first transcript, exon 10 was spliced to exon 13, and in the second transcript intron 11 was retained. The relative amount of mutant transcripts from 14 of the 22 subjects was determined by use of an RT-PCR-based method. Quantitation of the relative amounts of mutant transcripts for five missense mutations resulted in a mean value (+/-SD) of 52.8% (+/-4.55). In comparison, quantitation of the relative amounts of mutant transcripts for five nonsense mutations resulted in a mean value of 31.8% (+/-6.91). This value was significantly lower than the value of 54.2% (+/-2.38) obtained for nine healthy subjects (P < 0.0001). The relative amount of mutant transcripts for the 1705 + 1G-->T mutation was 36%. Thus, transcripts from alleles containing premature stop codons are present in reduced amounts, whereas transcripts from alleles containing missense mutations are present in normal amounts. These findings underscore the importance of determining how mutations affect mRNA structure and quantity in order to understand how mutations cause disease.

Alleles↗
PubMed1999

Multiple dysfunctions of two apolipoprotein A-I variants, apoA-I(R160L)Oslo and apoA-I(P165R), that are associated with hypoalphalipoproteinemia in heterozygous carriers.

ApoA-I(R160L)Oslo and apoA-I(P165R) are naturally occurring apolipoprotein (apo) A-I variants that are associated with low HDL-cholesterol in heterozygous carriers. We characterized the capacity of these variants to bind lipid, to activate lecithin:cholesterol acyltransferase (LCAT), and to promote efflux of biosynthetic cholesterol from porcine aortic smooth muscle cells (SMCs) or exogenous cholesterol from lipid-loaded mouse peritoneal macrophages. During cholate dialysis, normal apoA-I and both variants associated completely with dipalmitoylphosphatidylcholine (DPPC) and formed rLpA-I of identical size. However, both apoA-I(P165R) and apoA-I(R160L)Oslo showed a reduced capacity to clear a turbid emulsion of dimyristoylphosphatidylcholine (DMPC). Compared to normal apoA-I, the LCAT-cofactor activity of apoA-I(P165R) and apoA-I(R160L)Oslo as defined by the ratio of Vmax to appKm was reduced significantly by 62% and 29%, respectively (here and throughout the text, the apparent Km is given as Michaelis-Menten kinetics do not take particle binding into account and therefore would result in errors with an interfacial enzyme such as LCAT; Vmax estimates are not affected by this error). ApoA-I/DPPC complexes induced biphasic cholesterol efflux from SMCs with a fast and a slow efflux component. Compared to rLpA-I reconstituted with wild type apoA-I, rLpA-I with apoA-I(P165R) or apoA-I(R160L)Oslo were significantly less effective in promoting cholesterol efflux from SMCs in incubations of 10 min duration but equally effective in incubations of 6 h duration. Lipid-free apoA-I did not induce efflux of biosynthetic cholesterol from SMCs but induced hydrolysis of cholesteryl esters and cholesterol efflux from acetyl-LDL-loaded mouse peritoneal macrophages. In the lipid-free form, both apoA-I variants promoted normal cholesterol efflux from murine peritoneal macrophages. We conclude that amino acid residues arginine 160 and proline 165 of apoA-I contribute to the formation of a domain that is very important for initial lipid binding and contributes to LCAT-activation and promotion of initial cholesterol efflux but not to the stabilization of preformed rLpA-I.

Animals↗
PubMed1999

Phenotypic consequences of a deletion of exons 2 and 3 of the LDL receptor gene.

Screening for structural alterations of the low density lipoprotein (LDL) receptor gene by Southern blot analysis revealed an abnormal band pattern in one subject with a clinical diagnosis of homozygous familial hypercholesterolemia (FH). The molecular defect was further characterized by polymerase chain reaction and cDNA sequencing. These analyses identified a 4.8 kb in-frame deletion of exons 2 and 3, where exon 1 was spliced to exon 4. This deletion is expected to produce a receptor that has lost the two first cysteine-rich repeats of the ligand-binding domain. Previously published data of in vitro site-directed mutagenesis has shown that binding of LDL to such a receptor is reduced to 70% of normal. A mild phenotype in our FH homozygote is consistent with that observation. In contrast, heterozygotes carrying this deletion have a relatively more severe phenotype that is comparable to that of heterozygotes carrying a null-allele. A severe phenotype was also found in a compound heterozygote carrying this deletion. Possible mechanisms for this phenotypic variability are discussed.-Rødningen, O. K., S. Tonstad, J. D. Medh, D. A. Chappell, L. Ose, and T. P. Leren. Phenotypic consequences of a deletion of exons 2 and 3 of the LDL receptor gene.

Base Sequence↗
PubMed1998

Effects of a 9.6-kb deletion of the LDL receptor gene (FH Helsinki) on structure and levels of mRNA.

FH Helsinki is a deletion of the low-density lipoprotein receptor (LDLR) gene that deletes 9.6 kb from intron 15 to exon 18. Screening for mutant transcripts by Northern blot analysis from a patient heterozygous for FH Helsinki revealed two mutant transcripts. One was a transcript where the proximal part of intron 15 was retained in mRNA. The second was a transcript where exon 15 was spliced to nucleotide 4186 of exon 18. Thus, this transcript was generated using the normal donor splice site in intron 15, and a cryptic AG acceptor splice site in exon 18. Translation of the two mutant transcripts is predicted to give nonfunctional proteins, as both the membrane-spanning domain and the cytoplasmic domain of the receptor are deleted. Scanning of the autoradiograms showed that the amounts of each of the two mutant transcripts were approximately 10 times higher than that of the normal transcript in our heterozygous patient. The finding of higher levels of mutant transcripts was confirmed by an allele-specific transcript quantitation method, in which the amount of the two mutant transcripts together was approximately 5 times higher than the amount of the normal transcript. Deletion of destabilizing elements (AU-rich elements) by FH Helsinki are proposed to cause the increased levels of mutant transcripts.

Base Sequence↗
PubMed1998

Screening for mutations of the apolipoprotein B gene causing hypocholesterolemia.

In this study we have performed analyses of apolipoprotein (apo) B at both the protein and gene level to search for mutations of the apoB gene causing hypocholesterolemia among 71 Norwegian subjects. None of the subjects possessed apoB of abnormal molecular weight as determined by SDS-polyacrylamide gel electrophoresis of lipoproteins in the 1.025 g/ml-1.063 g/ml density range. Screening for mutations in exon 26 of the apoB gene by analysis of single-strand conformation polymorphisms followed by DNA sequencing, revealed seven point mutations of which one is a novel mutation. Five of the mutations were missense mutations and two were sense mutations. A group of 143 hypercholesterolemic, nonfamilial hypercholesterolemia subjects served as a control group for comparisons of gene frequencies. The only statistically significant finding was that mutation 8344T at codon 2712 was more common among those with hypocholesterolemia. This finding is in accord with previous reports.

Adult↗
PubMed1998

Carotid intima-media thickness and plaque in patients with familial hypercholesterolaemia mutations and control subjects.

BACKGROUND: In individuals with familial hypercholesterolaemia (FH), ultrasonographic measurement of carotid intima-media thickness (IMT) and plaque may provide a non-invasive assessment of cardiovascular risk. METHODS: We examined carotid artery IMT and its determinants in 79 non-smoking, normotensive, treated men and women with FH aged 26-46 years, and in 79 non-smoking, normotensive sex-, age- and body mass index-matched control subjects. FH was verified by molecular genetic analyses. The underlying mutation in the low-destiny lipoprotein receptor gene included a splice-site mutation, mutations predicted or shown to lead to class 2B mutations or other mutations that probably represent class I mutations (null alleles). RESULTS: The carotid bifurcation and common carotid artery IMT was increased in men with FH compared with control subjects (0.81 +/- 0.15 mm vs. 0.74 +/- 0.19 mm and 0.61 +/- 0.13 mm vs. 0.55 +/- 0.14 mm respectively; P < 0.05). The carotid bifurcation IMT was increased in women with FH compared with control subjects (0.74 +/- 0.17 vs. 0. 66 +/- 0.15; P = 0.005). More subjects with FH had carotid plaque (54% vs. 14%; P = 0.0001). In multivariate analysis, male gender, level of low-density lipoprotein-cholesterol, cholesterol-years score and xanthoma were associated with IMT and plaque in subjects with FH. FH subjects with class 2B mutations had lower cholesterol levels than subjects with mutations belonging to the other classes. They also had a tendency towards a decreased common carotid artery IMT. CONCLUSION: These findings confirm the importance of gender, xanthoma and lifetime cholesterol levels in relation to carotid atherosclerosis in FH. Whether the type of mutation causing FH modulates carotid artery IMT and plaque requires further study.

Adult↗
PubMed1997

[Diagnosis and treatment of severe hyperlipidemia].

Though severe hyperlipidaemia (total cholesterol level > or = 13 mmol/l in this study) is uncommon, it is important to make a precise diagnosis. We examined 57 patients with isolated severe hypercholesterolaemia. Of these, four were homozygotes for familial hypercholesterolaemia, 48 were heterozygotes for familial hypercholesterolacmia and one had sitosterolemia. The heterozygotes carried 15 different LDL receptor mutations, with no one mutation predominating. When the diagnosis is made, relatives should be given the opportunity to be tested. Combined severe hyperlipidaemia is usually due to a secondary cause, at our clinic, the most common cause is diabetes mellitus. The underlying disease should be treated first. However, many patients will require additional lipid-lowering drugs because the underlying disease may be associated with an increased risk of cardiovascular disease. With the exception of fish oil capsules, drugs that reduce serum triglyceride levels substantially are not registered in Norway at present.

Adult↗
PubMed1997

Flow cytometric measurement of low density lipoprotein receptor activity validated by DNA analysis in diagnosing heterozygous familial hypercholesterolemia.

We have evaluated whether low density lipoprotein (LDL) receptor activity of stimulated lymphocytes, as measured by an improved flow cytometric assay, may be used to diagnose familial hypercholesterolemia (FH). Cells were isolated from 75 children suspected from strict clinical criteria to be FH heterozygotes and from 29 normal children. DNA from the FH patients were also subjected to molecular genetic analysis of the LDL receptor gene in order to confirm the clinical diagnosis. A molecular genetic diagnosis of FH was obtained in 68 of the 75 patients; 67 of these had a low (below 70% of normal) receptor activity and 1 had a borderline (71%) activity. By contrast, 28 of the normal children showed a normal (above 80%) and 1 a borderline (78%) receptor activity. Of the 7 patients in whom no mutation in the LDL receptor gene was found, 4 showed a normal, 1 a borderline, and 2 showed a low activity. In summary, measurement of LDL receptor activity allowed us to separate between genetically diagnosed FH heterozygotes and healthy children. The combined use of LDL receptor activity measurements and molecular genetic analysis allows us both to diagnose and exclude FH in children suspected to suffer from this disease.

Child↗
PubMed1997

[Application of gene technology in the diagnosis of familial hypercholesterolemia].

Familial hypercholesterolaemia is an autosomal dominant disorder characterized by hypercholesterolaemia, xanthomas and premature coronary heart disease. Treatment of hypercholesterolemia is effective and consists of dietary changes and lipid lowering drugs. Only a minor proportion of familial hypercholesterolaemia patients are adequately treated, however. One explanation for this is assumed to be the relatively vague clinical diagnostic criteria applied. Because familial hypercholesterolaemia is caused by a mutation in the gene encoding the low density lipoprotein (LDL) receptor, mutation analysis of this gene could form the basis for specific diagnosis. 29 different mutations in the LDL receptor gene have been found to cause familial hypercholesterolaemia among Norwegian patients, and a total of 681 patients from 322 unrelated families have been provided with a molecular genetic diagnosis. We conclude that the use of molecular genetic analysis is feasible, and should be used clinically.

DNA Mutational Analysis↗
PubMed1997

[Is there a need for genetic/molecular diagnosis of familial hypercholesterolemia?].

Patients with familial hypercholesterolaemia have a significantly elevated risk of coronary heart disease. Accordingly, it is of crucial importance to diagnose and treat these patients before they contract premature coronary heart disease. At present, however, only a small proportion of familial hypercholesterolaemia patients are treated adequately. One main reason for this is probably the relatively vague clinical diagnostic criteria applied. We therefore advocate instead the use of molecular genetics to obtain a specific diagnosis by identifying the underlying genetic defect.

Female↗