J.A. Gevers Leuven

Lipoprotein(a): relation to other risk factors and genetic heritability. Results from a Dutch parent-twin study.

We measured plasma levels of lipoprotein(a) (Lp(a)) in a sample of 152 Dutch adolescent mono- and dizygotic twin pairs and their parents. The distribution of Lp(a) levels was skewed, with the highest frequencies at low levels and was similar for adult men and women and their children. The relationship of Lp(a) concentrations with other lipoprotein and apolipoprotein risk factors for coronary heart disease and with lathosterol, an indicator of whole-body cholesterol synthesis, was studied dependent on sex and generation. In mothers and children there was a small positive correlation between Lp(a) levels and plasma cholesterol and apolipoprotein (apo) B. In mothers and daughters there also was a correlation between Lp(a) and LDL cholesterol levels. No correlation was found between Lp(a) levels and plasma lathosterol, suggesting that there is no relationship between Lp(a) levels and cholesterol synthesis. Associations among family members, i.e. between monozygotic and dizygotic twins and between parents and offspring were used to study familial transmission of Lp(a) levels. Results showed that almost all of the variance in Lp(a) concentrations was accounted for by genetic heritability. A small, but significant, sex difference in heritability was observed, but heritabilities were the same in parents and offspring. Heritability estimates were 93% for females and 98% for males. No evidence was found for assortative mating or for the influence of a shared family environment. These results indicate that nearly all variance in Lp(a) concentrations that is not accounted for by the apo(a) size polymorphism, is also under genetic control.

Apolipoprotein E3-Leiden. A new variant of human apolipoprotein E associated with familial type III hyperlipoproteinemia.

SummaryA variant of apolipoprotein E, denoted apo E3-Leiden, has been identified in a 41-year-old male suffering from type III hyperlipoproteinemia with xanthomatosis. Apo E3-Leiden focus in the E3 position. In contrast with normal apo E3, apo E3-Leiden is defective in binding to the low density lipoprotein (LDL) receptor and does not contain cysteine as evaluated by cysteamine treatment of very low density lipoprotein followed by isoelectric focusing and conventional protein staining and by amino acid analysis. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, apo E3-Leiden displays an electrophoretic mobility intermediate to that of normal apo E3 and apo E2 (Arg158→Cys). The mother and four siblings of the proband also have apo E3-Leiden and hyperlipoproteinemia type III; three of them with xanthomatosis. Two siblings do not show apo E3-Leiden in their VLDL fraction and do not have hyperlipoproteinemia type III. In the VLDL fractions of all affected family members only the presence of apo E3-Leiden could be detected after cysteamine treatment and isoelectric focusing followed by conventional protein staining. However, isoelectric focusing of cysteaminetreated sera followed by immunoblotting, using anti-apo E antiserum as first antiserum, demonstrates the presence of low amounts of normal apo E3 in addition to apo E3-Leiden in serum of the affected family members. These results indicate that all affected family members are heterozygotes E3/E3-Leiden and suggest that in this family type III hyperlipoproteinemia is transmitted as a dominant trait.

Genetic analysis of sex and generation differences in plasma lipid, lipoprotein, and apolipoprotein levels in adolescent twins and their parents

In a sample of Dutch families consisting of parents aged 35–65 years and their twin offspring aged 14–21 years, a significant difference between generations was observed in phenotypic variances and in genetic heritabilities for plasma levels of total cholesterol, triglycerides, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol, and apolipoproteins (apo) A1, A2, B, and E. For all traits parents were more variable than their offspring. This increase in phenotypic variance was best explained by a genetic model in which individual specific environmental variance increased with increasing age. Genetic variance was the same across generations for nearly all traits except triglycerides and apoE, for which a decrease in genetic variance was observed. This model led to large intergenerational differences in genetic heritabilities. Heritabilities for children were between 65 and 87%, while heritabilities for their parents were between 10 and 50%. No evidence was found for effects of a shared family environment.

Apolipoprotein E*3-Leiden allele results from a partial gene duplication in exon 4

The apolipoprotein E3-Leiden variant has been shown to be associated with familial dysbetalipoproteinemia (FD) in a dominant manner (Havekes et al., Hum Genet 1986;73:157-163). Applying the polymerase chain reaction technique, we have cloned and sequenced relevant parts of both APOE alleles of the original proband. In exon 4 of the E*3-Leiden allele a partial gene duplication encompassing 21 nucleotides was found, leading to a tandem repeat of the codons 120-126 or 121-127. Using an E3-Leiden mutation specific oligonucleotide probe, the same mutation was found in two additional independently ascertained FD patients with an E3E3 phenotype based on isoelectric focusing. The E*3-Leiden mutation will be useful in the elucidation of the etiology of dominantly inherited forms of FD.

Dietary counselling effectively improves lipid levels in patients with endogenous hypertriglyceridemia: emphasis on weight reduction and alcohol limitation

Objective: To evaluate the short-term effect of dietary counselling in patients with endogenous hypertriglyceridemia and evaluate the effects of advised nutrient changes.Design: A prospective dietary intervention study in patients with endogenous hypertriglyceridemia from January 1st 1988 to December 31st 1996 according to the Dutch guidelines for a healthy diet. Before and after the dietary intervention period of 12 weeks, 24 h food recalls were used to assess dietary intake and macronutrient composition. Effectiveness was evaluated by assessment of body weight, serum lipids, lipoproteins and insulin resistance parameters.Setting: Leiden outpatient Lipid Clinic.Subjects: Forty-five newly diagnosed, untreated patients with endogenous hypertriglyceridemia.Results: A significant reduction in energy intake and body weight as well as changes in macronutrient composition were observed. Total serum triacylglycerol and cholesterol levels decreased by 31% and 15%, respectively. No effects were observed on serum glucose and insulin levels. Weight reduction was significantly correlated with reduction of total plasma triacylglycerol levels and inversely correlated with changes in HDL cholesterol levels. Of all nutrients assessed, only reduction of alcohol intake correlated with improvement of total serum triacylglycerol.Conclusions: Short-term dietary counselling in patients with endogenous hypertriglyceridemia can effectively improve serum lipid and lipoprotein levels. With regard to the advised nutrient changes, weight loss and limitation of alcohol intake prove to be the best predictors of triacylglycerol reduction.

Absence of mutations in the promoter region of the low density lipoprotein receptor gene in a large number of familial hypercholesterolaemia patients as revealed by denaturing gradient gel electrophoresis.

SummaryDenaturing gradient gel electrophoresis (DGGE) was used in combination with the polymerase chain reaction (PCR) to detect sequence variations in the promoter region of the low density lipoprotein receptor (LDLR) gene. On the basis of calculated predictive melting properties we designed primers to amplify a 447-bp fragment of the promoter region from position-512 to -66, containing previously identified regulatory sequences. Using a primer with a GC-clamp in combination with restriction enzyme digestion, two melting domains could be analysed simultaneously. By oligonucleotide-directed mutagenesis artificial mutants were generated to optimize the conditions and to test the sensitivity of the method. All mutants were readily detected by electrophoresis in a 9% polyacrylamide gel containing a 10%–60% linear denaturing gradient. Using this method, we analysed DNA samples of 350 heterozygous familial hypercholesterolaemia (FH) patients. No mutations were detected, suggesting that mutations in the regulatory elements of the promoter sequence do not play a significant role in the etiology of FH.

The Effect of Cyclandelate on Cholesterol Metabolism in Patients with Familial Hypercholesterolaemia

SummaryHeterozygous familial hypercholesterolaemia (HtFH) is associated with an increased risk of coronary artery disease. Prevention is possible by increasing the number of functioning receptors of low density lipoproteins (LDLs) in the liver. This is partly achieved by treatment with bile acid sequestrants, such as cholestyramine, but the effect is limited because of a concomitant increase in cholesterol synthesis.It was the purpose of this study to determine whether the increase in cholesterol synthesis could be influenced by treatment with cyclandelate, since it is known that cyclandelate inhibits cholesterol synthesis in rats.Ten patients received cyclandelate (3.2g daily in 2 doses) or placebo in a double- blind crossover study, with each treatment period of 3 months’ duration. During these periods, treatment with cholestyramine (16g daily) was continued. No evidence was found of inhibition of cholesterol synthesis by cyclandelate, as indicated by the serum concentration of the cholesterol precursor, lanosterol, which remained unchanged. Neither the serum concentration of LDL, nor those of high density lipoprotein (HDL) cholesterol, apolipoprotein B, A- I or A- II, were affected. Thus, it can be concluded that treatment with cyclandelate was not effective in lowering serum cholesterol concentrations in patients with familial hypercholesterolaemia who received concomitant cholestyramine therapy.RésuméL’hypercholestérolémie familiale hétérozygote est responsable d’un risque accru de coronaropathie, pouvant être évité par l’augmentation du nombre des récepteurs hépatiques des lipoprotéines de faible densité (LDL). Cette prévention peut être en partie obtenue à l’aide d’un traitement par des fixateurs des acides biliaires, comme la cholestyramine, mais cet effet est néanmoins limité en raison de l’augmentation concomitante de la synthèse du cholestérol.Le cyclandélate inhibant la synthèse du cholestérol chez le rat, le but de la présente étude était de déterminer si ce produit possède une quelconque influence sur l’augmentation de cette synthèse chez l’homme.Au cours d’une étude croisée en double-insu, chaque patient a reçu quotidiennement 3,2 g de cyclandélate en 2 prises ou un placebo, chaque période de traitement durant 3 mois. L’administration de 16 g quotidiens de cholestyramine s’est poursuivie pendant toute la durée de l’étude. Les concentrations sériques du lanostérol, précurseur du cholestérol, étant restées inchangées, cette étude n’a pas pu mettre en évidence une quelconque inhibition de la synthèse du cholestérol par la cyclandélate. Les taux sériques de LDL n’ont pas été modifiés par le traitement; il en est allé de même pour les taux sériques du cholestérol des lipoprotéines de haute densité (HDL) et des apolipoprotéines B, AI et AH. Il faut donc conclure que le cyclandélate n’a pas abaissé de façon efficace la cholestérolémie chez des patients atteints d’hypercholestérolémie familiale et recevant un traitement concomitant par la cholestyr- amine.RiassuntoLa ipercolesterolemia (HtFH) familiäre eterozigotica è associata ad un aumentato rischio di malattia coronarica. Incrementando il numéro dei recettori funzionali délie lipoprotéine di bassa densità (LDL) nel fegato è possibile la prevenzione. Questa si ottiene in parte mediante trattamento con sequestranti dell’acido biliare quali colestiramina, ma l’effetto è limitato perché si ha un concomitante aumento délia sintesi del colesterolo.Lo scopo di questo studio era di determinare se Vawnento nella sintesi del colesterolo potesse essere influenzato dal trattamento con ciclandelato dato che è noto che il ciclandelato inibisce la sintesi del colesterolo nel ratto.Dieci pazienti ricevettero ciclandelato o placebo alla dose di 3,2 g al giorno, suddivisa in due volte, in uno studio di crossover in doppio cieco; ogni trattamento durò tre mesi. Durante questi periodi il trattamento con colestiramina venne continuato alla dose quotidiana di 16 g. Non si evidenziò inibizione alla sintesi del colesterolo da parte del ciclandelato come indicato dalla concentrazione del precursore del colesterolo (il lanosterolo), che rimase inalterata. Nonfurono influenzate nemmeno la concentrazione nel siero di LDL, né quella delle lipoproteine di alta densità (HDL), colesterolo, apolipoproteina B, A-I o A-II Pertanto si può concludere che il trattamento con ciclandelato non è stato efficace nel ridurre la concen- trazione del colesterolo nel siero di pazienti con ipercolesterolemia familiäre che ricevevano una terapia concomitante di colestiramina.SamenvattingHeterozygote familiale hypercholesterolemie (HtFH) geeft een verhoogd risico op coronair vaatlijden. Preventie is mogelijk door het aantal functionerende LDL-receptoren in de lever te verhogen. Dat wordt gedeeltelijk bereikt door behandeling met galzuur bindende Stoffen zoals cholestyramine, maar het effect is beperkt ten gevolge van een gelijktijdige stijging van de cholesterolsynthese.Het doel van deze studie was na te gaan of de stijging van de cholesterolsynthese kon worden beïnvloed door behandeling met cyclandelaat, aangezien men weet dat cyclandelaat de cholesterolsynthese bij ratten remt.Tien patiënten kregen tijdens een dubbelblinde, gekruiste studie 3,2 g cyclandelaat in 2 dosissen of placebo per dag. Elke behandelingsperiode duurde 3 maanden. Tijdens die pe- rioden werd de behandeling met 16 g cholestyramine per dag voortgezet. Er kon geen rem- ming van de cholesterolsynthese door cyclandelaat worden vastgesteld, zoals bleek uit de serumconcentratie van de cholesterolprecursor lanosterol, die onveranderd bleef Noch de serumconcentratie van LDL, noch die van HDL of de apolipoproteïnen B, A-I of A-Il werd beïnvloed. De conclusie was daarom dat behandeling met cyclandelaat de serumcholesterol concentraties niet verlaagt bij patiënten die tegelijkertijd met cholestyramine behandeld worden.