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Biomedical subjects

T Bruin

Publications and source records attributed to T Bruin.

At least 19 recordsLinked to original sources
PubMed1999

Influence of repetitive finger puncturing on skin perfusion and capillary blood analysis in patients with diabetes mellitus.

BACKGROUND: Frequent puncturing of fingers to check blood glucose in patients with type 1 diabetes might alter skin perfusion and, hence, influence the representativeness of the blood sample. We investigated the influence of repetitive puncturing on skin microcirculatory perfusion using laser Doppler fluxmetry and on the preanalytical phase of capillary blood analysis for small molecules (glucose) and large particles (cholesterol). METHODS: In 49 patients with long-standing (mean, 21 years) type 1 diabetes, with a mean puncture frequency of three times daily for a mean duration of 13 years, laser Doppler skin perfusion was measured in a finger at a frequently punctured site and compared with a similar site of another finger of the same hand, which was never punctured. In the supine position with the hand level with the heart, resting flux (RF), peak flux (PF), and the microcirculatory reserve capacity (MRC; PF - RF) were assessed. Subsequently, blood samples for capillary whole blood glucose and cholesterol analyses were taken from the same sites. RESULTS: No significant differences were found between the puncture and control sites in mean RF (2.3 vs 2.0 V; P = 0.14, paired-samples t-test), PF (3.3 vs 3.1 V; P = 0.24), MRC (1.0 vs 1.0 V; P = 0.65), glucose (10.2 vs 10.2 mmol/L; P = 0.69), or cholesterol (5.1 vs 5.2 mmol/L; P = 0.26). Power calculation for a RF of 2.0 V and the SD and n of this study indicate a power (beta) of 80% to detect a 25% change in RF at P <0.05. CONCLUSIONS: Repetitive finger puncturing in diabetics appears not to injure local skin microcirculatory perfusion nor to influence results of capillary blood analysis for glucose and cholesterol.

Adult↗
PubMed1998

Ile225Thr loop mutation in the lipoprotein lipase (LPL) gene is a de novo event.

Mutations in the lipoprotein lipase (LPL) gene are the most important cause of familial chylomicronemia with over 70 mutations being recorded to date. Thus far de novo mutations have not been described. Here we report on the molecular analysis of the family of a patient previously reported to be LPL deficient on the basis of compound heterozygosity for the Arg243His and Ile225Thr mutations, the latter being the first and only mutation identified in the loop region of LPL. Both parents of the propositus were screened for the presence of these two mutations to confirm their status as obligate heterozygotes and to determine the mutation allocation. Although paternal inheritance of the Arg243His allele could be established, maternal DNA did not show carrier status for the Ile225Thr substitution. An examination of maternity, using LPL restriction fragment length polymorphisms four polymorphic CA repeats and ApoE genotypes, was consistent with correct biological parentage for the propositus. Therefore, we conclude that the Ile225Thr mutation constitutes a de novo event, the first to be reported in the LPL gene.

Adult↗
PubMed1997

Genetic variant showing a positive interaction with beta-blocking agents with a beneficial influence on lipoprotein lipase activity, HDL cholesterol, and triglyceride levels in coronary artery disease patients. The Ser447-stop substitution in the lipoprotein lipase gene. REGRESS Study Group.

BACKGROUND: Lipoprotein lipase (LPL) is the rate-limiting enzyme in the lipolysis of triglyceride-rich lipoproteins, and the gene coding for LPL is therefore a candidate gene in atherogenesis. We previously demonstrated that two amino acid substitutions in LPL, the Asn291-Ser and the Asp9-Asn, are associated with elevated triglycerides and lower HDL cholesterol and are present with greater frequency in coronary artery disease (CAD) patients than in normolipidemic control subjects. Conversely, a third frequent mutation in this gene, the Ser447-Stop, is reported by some investigators to underlie higher HDL cholesterol levels and would represent a beneficial genetic variant in lipoprotein metabolism. We therefore sought conclusive evidence for these allegations by investigating the effects of the LPL Ser447-Stop mutation on LPL and hepatic lipase (HL) activity, HDL cholesterol, and triglycerides in a large group of CAD patients (n = 820) with normal to mildly elevated total and LDL cholesterol levels. METHODS AND RESULTS: Carriers of the Ser447-Stop allele (heterozygotes and homozygotes) had significantly higher postheparin LPL activity (P = .034), normal postheparin HL activity (P = .453), higher HDL cholesterol levels (P = .013), and lower triglyceride levels (P = .044) than noncarriers. The influence of the Ser447-Stop allele on LPL activity was pronounced in patients using beta-blockers (P = .042) and not significant in those not using them (P = .881), suggesting a gene-environment interaction between the Ser447-Stop mutation and beta-blockers. CONCLUSIONS: We conclude that the LPL Ser447-Stop mutation has a significant positive effect on LPL activity and HDL cholesterol and triglyceride levels and that certain subgroups of CAD patients carrying the Ser447-Stop mutation will have less adverse metabolic effects when placed on beta-blockers. The LPL Ser447-Stop mutation therefore should have a protective effect against the development of atherosclerosis and subsequent CAD.

Adrenergic beta-Antagonists↗
PubMed1997

Mutation of tryptophan residues in lipoprotein lipase. Effects on stability, immunoreactivity, and catalytic properties.

Previous studies had pointed to an important function of a putative exposed loop in the C-terminal domain of lipoprotein lipase for activity against emulsified lipid substrates. This loop contains 3 tryptophan residues (Trp390, Trp393, and Trp394). We have expressed and characterized lipase mutants with tryptophan to alanine substitutions at positions 55, 114, 382, 390, 393, and 394 and a double mutant at residues 393 and 394. The substitutions in the N-terminal domain (W55A and W114A) led to poor expression of completely inactive lipase variants. Heparin-Sepharose chromatography showed that mutant W114A eluted at the same salt concentration as inactive wild-type monomers, indicating that this substitution prevented subunit interaction or led to an unstable dimer. In contrast, all mutants in the C-terminal domain were expressed as mixtures of monomers and dimers similarly to the wild-type. The dimers displayed at least some catalytic activity and had the same apparent heparin affinity as the active wild-type dimers. The mutants W390A, W393A, W394A, and W393A/W394A had decreased reactivity with the monoclonal antibody 5D2, indicating that the 5D2 epitope is longer than was reported earlier, or that conformational changes affecting the epitope had occurred. The mutants W390A, W393A, W394A, and W393A/W394A had decreased catalytic activity against a synthetic lipid emulsion of long-chain triacylglycerols (IntralipidR) and in particular against rat lymph chylomicrons. The most pronounced decrease of activity was found for the double mutant W393A/W394A which retained only 6% of the activity of the wild-type lipase, while 70% of the activity against water-soluble tributyrylglycerol was retained. In the case of chylomicrons also the affinity for the substrate particles was lowered, as indicated by severalfold higher apparent Km values. This effect was less prominent with the synthetic lipid emulsion. We conclude that the tryptophan cluster Trp390-Trp393-Trp394 contributes to binding of lipoprotein lipase to lipid/water interfaces. Utilizing different lipid substrates in different physical states, we have demonstrated that the tryptophan residues in the C-terminal domain may have a role also in the productive orientation of the enzyme at the lipid/water interface.

Amino Acid Sequence↗
PubMed1997

A single Ser259Arg mutation in the gene for lipoprotein lipase causes chylomicronemia in Moroccans of Berber ancestry.

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglyceride-rich lipoproteins. Numerous LPL gene mutations have been described as a cause of familial chylomicronemia in various populations. In general, allelic heterogeneity is observed in LPL deficiency in different populations. However, a founder effect has been reported in certain populations, such as French Canadians. Although familial chylomicronemia is observed in Morocco, the molecular basis for the disease remains unknown. Here, we report two unrelated Moroccan families of Berber ancestry, ascertained independently in Holland and France. In both probands, familial chylomicronemia manifested in infancy and was complicated with acute pancreatitis at age 2 years. Both probands were homozygous for a Ser259Arg mutation, which results in the absence of LPL catalytic activity both in vivo and in vitro. In heterozygous relatives, a partial decrease in plasma LPL activity was observed, sometimes associated with combined hyperlipidemia. This mutation previously unreported in other populations segregated on an identical haplotype, rarely observed in Caucasians, in both families. Therefore, LPL deficiency is a cause of familial chylomicronemia in Morocco and may result from a founder effect in patients of Berber ancestry.

Adolescent↗
PubMed1997

Ser447stop mutation in lipoprotein lipase is associated with elevated HDL cholesterol levels in normolipidemic males.

This report describes the association between a frequent mutation in the lipoprotein lipase (LPL) gene and HDL cholesterol levels. It concerns a previously described defect that predicts a premature truncation of the LPL protein (447stop). We determined the frequency of this mutation in three groups of healthy men with low-, middle-, and upper-decile HDL cholesterol. The number of carriers of the 447stop allele was significantly greater in the high HDL group than in either the groups with normal HDL (P = .017) or low HDL (P < .0001). Additional functional assessment of this mutation did not reveal distinct differences between wild-type LPL and the LPL447stop protein. In conclusion, we have shown that the 447stop mutation is associated with increased HDL cholesterol in healthy Dutch males, although the underlying mechanism remains to be elucidated. Because HDL cholesterol is strongly inversely related with CAD, this genotype might be of potential benefit to its carriers.

Adult↗
PubMed1996

The Asp9 Asn mutation in the lipoprotein lipase gene is associated with increased progression of coronary atherosclerosis. REGRESS Study Group, Interuniversity Cardiology Institute, Utrecht, The Netherlands. Regression Growth Evaluation Statin Study.

BACKGROUND: Many patients suffering from premature coronary artery disease report a family history for such events. A mutation in a particular gene, which confers susceptibility for atherosclerosis, will be found more frequently in individuals suffering from coronary atherosclerosis than in the general population. We have recently reported the identification of an Asp9 Asn substitution in the lipoprotein lipase (LPL) enzyme. We analyzed the impact of this mutation on the progression of coronary atherosclerosis and the effect of pravastatin in both carriers and noncarriers. METHODS AND RESULTS: All patients were enrolled in the quantitative coronary angiographic clinical trial REGRESS, which studied the impact of pravastatin therapy on coronary atherosclerosis. The Asp9 Asn mutation was identified in 38 of 819 (4.8%) patients. Carriers of the mutation more often had a positive family history of cardiovascular disease and lower HDL cholesterol levels than noncarriers. In the placebo group, carriers showed more progression of coronary atherosclerosis than noncarriers: mean reduction of the minimum obstruction diameter of -0.25 mm versus -0.12 mm (P = .029) and increase of percentage diameter stenosis of 6.4% versus 1.4% (P = .004). Moreover, the adjusted relative risk for a clinical event for carriers was calculated at 2.16 (95% CI, 1.09 to 4.29; P = .027). Although the lipid-lowering effect of pravastatin was attenuated in carriers, it appeared that these patients showed a response similar to noncarriers in terms of less progression of atherosclerosis and event-free survival. CONCLUSIONS: This study shows that heterozygosity for a mutation in the LPL gene, which causes only subtle changes in fasting plasma lipids, may promote the progression of coronary atherosclerosis and diminish clinical event-free survival.

Amino Acid Sequence↗
PubMed1996

Compound heterozygosity for a known and a novel defect in the lipoprotein lipase gene (Asp250-->Asn; Ser251-->Cys) resulting in lipoprotein lipase (LPL) deficiency.

Two missense mutations in exon 6 of the LPL gene were identified on separate alleles in a Dutch patient with lipoprotein lipase (LPL) deficiency. The first mutation is a G1003-->A transition resulting in a D250N mutation, which has been shown previously to result in a catalytically defective protein in patients of French-Canadian ancestry. The second mutation, a C to G transition at nucleotide 1007, predicts a S251C residue change in the highly conserved region of LPL surrounding the loop structure the covers the catalytic triad. This mutation constitutes a novel defect among LPL gene mutations reported so far. Site-directed mutagenesis experiments provide in-vitro evidence for the complete loss of LPL activity resulting from this latter missense mutation. The G1003-->A nucleotide substitution underlying the Asp250 mutation deletes a TaqI endonuclease recognition site and the C1007-->G change that leads to the S251C alteration abolishes a HinfI recognition site. This will facilitate rapid screening for these mutations in LPL-deficient patients.

Adolescent↗
PubMed1995

A frequently occurring mutation in the lipoprotein lipase gene (Asn291Ser) contributes to the expression of familial combined hyperlipidemia.

We performed denaturing gradient gel electrophoresis (DGGE) of exons 4, 5, 6 and their exon-intron boundaries of the LPL-gene in 169 unrelated male patients suffering from familial combined hyperlipidemia (FCH). Twenty patients were found to carry a nucleotide substitution in exon 6. Sequence and PCR/digestion analysis revealed one common mutation (Asn291Ser) in all these cases. This mutation was talso present in 215 male controls, albeit at a lower frequency than in FCH patients (10/215 = 4.6% vs. 20/169 = 11.8%; p < 0.02). Analysis of lipid, lipoprotein and apolipoprotein levels demonstrated an association between the presence of this Asn291Ser substitution and decreased HDL-cholesterol (0.94 +/- 0.31 vs. 1.12 +/- 0.26 mmol/l; p < 0.04) in our controls. FCH patients carrying this mutation showed decreased HDL-cholesterol (0.75 +/- 0.16 vs. 0.95 +/- 0.36 mmol/l; p = 0.05) and increased triglyceride levels (5.96 +/- 4.12 vs. 3.48 +/- 1.78 mmol/l; p < 0.005) compared to non-carriers. The high triglyceride and low HDL-cholesterol phenotype in carriers of this substitution was most obvious when BMI exceeded 27 kg/m2. Our study of male FCH patients revealed the presence of a common mutation in the LPL-gene that is associated with lipoprotein abnormalities, indicating that defective LPL is at least one of the factors contributing to the FCH-phenotype.

Asparagine↗
PubMed1995

A common variant in the gene for lipoprotein lipase (Asp9-->Asn). Functional implications and prevalence in normal and hyperlipidemic subjects.

Subjects with combined hyperlipidemia (CHL) were screened for mutations in the lipoprotein lipase (LPL) gene by single-strand conformational polymorphism, and a previously reported G-->A DNA sequence change in exon 2, causing substitution of Asp by Asn at position 9, was identified in 2 individuals. Because this substitution destroys a recognition site for Taq I, pooling of DNA samples, amplification, and digest with Taq I allowed the rapid screening of 1563 healthy individuals and patients of Dutch, Swedish, English, and Scottish origin. In the general populations of all four countries, healthy carriers of the mutation were detected at a frequency of 1.6% to 4.4% (mean, 3.0%; 95% confidence interval, 2.0% to 4.0%). The frequency of carriers was roughly twice as high (range, 4.0% to 9.8%) in selected patients with CHL or type IV hyperlipoproteinemia or in subjects with angiographically assessed atherosclerosis; the frequency was consistently higher in each patient group compared with its matched control group. In 773 healthy men from two general practices in the United Kingdom, 25 carriers and 2 homozygotes for the mutation were identified. In these 27, plasma triglyceride but not plasma cholesterol levels were significantly higher than in noncarriers (2.25 versus 1.82 mmol/L, P < .02), and this difference was maintained in three subsequent annual measurements. Postheparin LPL activity data were available for some carriers and for 7 of 9 individuals from the patient groups, and 6 of 6 individuals from the control groups had LPL activity that was lower than the respective group mean. In vitro mutagenesis and transient expression in COS cells showed that compared with the LPL-Asp9 construct, LPL-Asn9 activity and mass were reduced by 20% to 30% in the culture media. Overall however, LPL-Asn9 had only slightly reduced specific activity (by 18%). Thus, although the precise mechanism of the effect is unclear, the data strongly suggest that the LPL-Asn9 variant is associated with and may play a direct role in predisposing carriers to develop hypertriglyceridemia.

Adult↗
PubMed1994

The C-terminus of lipoprotein lipase is essential for biological function but contains no domain for glycosylphosphatidylinositol anchoring.

In this study we present evidence that the C-terminus of lipoprotein lipase contains no glycosylphosphatidylinositol addition signal and is therefore not a glycosylphosphatidylinositol-anchored protein. Furthermore, we present additional evidence that the C-terminus of lipoprotein lipase is essential for biological function. Flow cytometric analysis and enzyme-activity monitoring experiments revealed no pool of lipoprotein lipase releasable by phosphatidylinositol-specific phospholipase present on the membrane of COS cells transfected with the human lipoprotein lipase gene while, in contrast, a heparin-releasable pool could be demonstrated. [14C]Ethanolamine, a constituent of the glycosylphosphatidylinositol anchor, was not incorporated into lipoprotein lipase during metabolic labeling. C-terminal deletion mutants were constructed and expressed in COS cells to investigate the presence of glycosylphosphatidylinositol addition signal on the C-terminus of human lipoprotein lipase (LPL). The specific activities of the mutants M442 [des-(Leu443-Gly448)-LPL] and M437 [des-(Cys438-Gly448)-LPL] were 78% and 59%, respectively, less than the wild type, while the M432 mutant [des-(Ala433-Gly449)-LPL] was catalytically inactive. Determination of the stability of the mutants revealed a decreased stability of the M437, compared with wild-type, whereas M442 showed the same stability. Flow cytometric analysis showed sustained membrane expression for all mutants including the inactive M432 mutant. These results suggest that the C-terminus of lipoprotein lipase is essential for maintaining intact catalytic activity but is not involved in any posttranslational proteolytic processing, including cleavage of a glycosylphosphatidylinositol addition signal. We therefore conclude that membrane-binding of the lipase is not mediated by such anchoring.

Amino Acid Sequence↗
PubMed1994

Recurrent missense mutations at the first and second base of codon Arg243 in human lipoprotein lipase in patients of different ancestries.

Mutations in the lipoprotein lipase (LPL) gene are the most common cause of familial chylomicronemia. Here we define the molecular basis of LPL deficiency in four patients of German, French, Dutch, and Chinese descent. We show that two of the probands of Dutch and Chinese origin have a previously described Arg243His mutation while the patients of German and French descent have a novel Arg243Cys substitution in their LPL gene. Haplotype analysis is in favour of two separate origins for the Arg243Cys substitution which together with the Arg243His mutation would implicate three recurrent mutations involving the first and second nucleotides of the codon encoding Arg243 of the LPL gene. The recurrent mutations affecting the first and second nucleotide of CGC coding for the normal Arg residue are support for the high mutability of CpG dinucleotides within the LPL gene.

Adult↗
PubMed1994

Homozygosity for a mutation in the lipoprotein lipase gene (Gly139-->Ser) causes chylomicronaemia in a boy of Spanish descent.

The enzyme lipoprotein lipase (LPL) plays a crucial role in triglyceride metabolism through catalysis of triglyceride-rich chylomicrons and very low density lipoproteins. Primary LPL deficiency manifests with chylomicronaemia and is caused by mutations in the LPL gene. In this paper we report a novel molecular defect (G670-->A) in exon 4 of the LPL gene, resulting in a substitution of serine for glycine at position 139 in the mature protein. We identified homozygosity for this mutation in a boy of Spanish descent. In vitro mutagenesis provided formal proof that this missense mutation completely abolishes LPL function and therefore is the cause of LPL deficiency.

Adolescent↗
PubMed1994

Apolipoprotein CII-Padova (Tyr37-->stop) as a cause of chylomicronaemia in an Italian kindred from Siculiana.

In this paper we report on the molecular defect underlying apolipoprotein CII (apoCII) deficiency in an Italian kindred. ApoCII serves as cofactor for lipoprotein lipase (LPL) in triglyceride hydrolysis of chylomicrons and very low density lipoproteins. Homozygous apoCII deficiency manifests with type I hyperlipoproteinaemia and is a rare disorder of lipoprotein metabolism. Until now, only 10 kindreds with apoCII deficiency have been published and all underlying mutations were unique. The proband was the offspring of a consanguineous mating. Sequencing of cloned DNA from the proband presented in this report showed homozygosity for a C-->A substitution at position 3002 in the apoCII gene, resulting in the introduction of a premature stop codon at residue 37 of the mature apoCII protein. Therefore, a truncated apoCII is synthesised, lacking the part of the apolipoprotein that activates LPL. This mutation has previously been described in another Italian family and is known as apoCIIPadova. We propose that apoCIIPadova is a frequent cause of apoCII deficiency in persons of Italian descent.

Abdominal Pain↗
PubMed1994

A compound heterozygote for lipoprotein lipase deficiency, Val69-->Leu and Gly188-->Glu: correlation between in vitro LPL activity and clinical expression.

We analyzed the molecular defects in the lipoprotein lipase gene of a patient with type I hyperlipidemia suffering from recurrent pancreatitis, indicative for lipoprotein lipase deficiency. Postheparin lipoprotein lipase activity in the patient was decreased by 70%. Direct genomic sequencing revealed compound heterozygosity for two mutation: the well-known Gly188-->Glu and a new Val69-->Leu substitution. Val69 is situated in a conserved hydrophobic region of the lipoprotein lipase protein, and the substitution with leucine gives rise to a 80% decrease in specific catalytic activity, as supported by site-directed mutagenesis experiments, followed by expression in COS-cells. The combination of both defects in the lipoprotein lipase gene was incidentally associated with severe clinical expression of disease, and triglyceride levels of more than 30 mmol/l were measured. In our patient, triglyceride levels wer usually below 10 mmol/l. We, therefore, postulate that the residual LPL activity in our patient is usually sufficient to keep the triglyceride level within bounds and expression of disease occurred only when conditions such as alcohol abuse or poor compliance to diet were present.

Adult↗
PubMed1993

Recurrent pancreatitis and chylomicronemia in an extended Dutch kindred is caused by a Gly154-->Ser substitution in lipoprotein lipase.

We report the molecular basis of familial chylomicronemia and recurrent pancreatitis in five members of a large Dutch family. All patients had normal plasma hepatic lipase and apoC-II levels, but absent lipoprotein lipase (LPL) catalytic activity and low LPL mass in postheparin plasma. The mutation in the LPL gene was characterized as a G715-->A substitution in the last nucleotide of exon 4, resulting in a substitution of Ser for Gly154. PCR amplification of exons 4 + 5 from the patients' mRNA, followed by direct sequencing, revealed normal splicing of intron 4. The mutation creates a BfaI restriction site that allows rapid screening of family members for the mutation. Reproduction of this mutation in LPL-cDNA by site-directed mutagenesis, followed by transient expression in COS-B cells, revealed production of a catalytically inactive enzyme. The Gly154-->Ser substitution appears in a conserved beta-sheet region, in close proximity to Asp156, which is part of the catalytic triad. These studies show that changes to residues close to Asp156 can have profound effects on catalytic activity of LPL.

Adult↗
PubMed1993

The familial hyperchylomicronaemia syndrome.

The familial hyperchylomicronaemia syndrome is a hereditary disorder of lipoprotein metabolism caused by lipoprotein lipase (LPL) deficiency, apolipoprotein(apo) CII deficiency or LPL inhibition. This syndrome, which is characterized by hyperchylomicronaemia, attacks of epigastric pain, recurrent pancreatitis and the presence of eruptive xanthomas, may ultimately lead to necrotizing pancreatitis or pancreatic insufficiency. Treatment consists of lifelong adherence to a low-fat diet to prevent hyperchylomicronaemia and its sequelae. We describe here the clinical course of a patient with acute pancreatitis due to hyperchylomicronaemia based on hereditary LPL deficiency. The different causes of the familial hyperchylomicronaemia syndrome and its therapy will be discussed and an update is presented of our knowledge concerning the basic molecular defects of this hereditary disorder.

Apolipoprotein C-II↗