A PCSK9 variant and familial combined hyperlipidaemia.

BACKGROUND: Our discovery in 2003 of the first mutations of PCSK9 gene causing autosomal dominant hypercholesterolaemia (ADH) shed light on an unknown factor that strongly influences the level of circulating low density lipoprotein cholesterol (LDL-C). PCSK9 gain of function mutations cause hypercholesterolaemia by a reduction of LDL receptor levels, while PCSK9 loss of function variants are associated with a reduction of LDL-C values and a decreased risk of coronary heart disease. METHODS AND RESULTS: We report an insertion of two leucines (p.L21tri also designated p.L15_L16ins2L) in the leucine stretch of the signal peptide of PCSK9 that is found in two of 25 families with familial combined hyperlipidaemia (FCHL). This mutant is associated with high total cholesterol and LDL-C values in these families and is found also in a patient with familial hypercholesterolaemia and her father. CONCLUSION: PCSK9 variants might contribute to FCHL phenotype and are to be taken into consideration in the study of this complex and multigenic disease with other genes implicated in dyslipidaemia.

Prevalence, risk factors and outcome of hyperlactataemia in HIV-infected patients.

OBJECTIVE: We describe the prevalence, risk factors and outcome of hyperlactataemia (HL) in a cohort of 140 HIV-infected patients. PATIENTS AND METHODS: Patients were enrolled consecutively within a 3-month period (July to September 1999) and followed until 31 October 2000. One hundred and forty HIV-infected patients had venous plasma lactate levels measured. HL was defined at baseline by two consecutive lactate levels > 2.1 mmol/L (upper limit of normal). We compared baseline demographic characteristics, immuno-virological parameters, antiretroviral therapy and outcome between patients with HL (cases) or without HL (controls). We described the clinical features of patients with HL. RESULTS: Among 129 patients included in the analysis, HL was found in 11 patients (8.5%), all of whom were receiving nucleoside reverse transcriptase inhibitors (NRTIs). Cases were more likely than controls to receive didanosine or stavudine (82% vs. 19%, P= 2.7 x 10(-6) and 82% vs. 48%, P= 0.03, respectively). Only 4/11 cases (36%) had symptoms consistent with HL. After a median follow-up of 15 months, lactate level returned to normal in all three patients who discontinued NRTIs, but in only 2/8 patients who did not (P = 0.06). Only one case experienced lactic acidosis and died during follow-up. Mortality rate was similar in cases and controls. CONCLUSION: HL is associated with NRTI use, in particular didanosine and stavudine, and discontinuation of NRTIs seems to be associated with rapid resolution of HL. Lactic acidosis remains rare and the long-term outcome of patients with HL does not seem to be poorer than controls.

Exercise training-induced triglyceride lowering negatively correlates with DHEA levels in men with type 2 diabetes.

OBJECTIVE: To investigate the effect of an exercise training program on lipid profile in correlation with DHEA level and body weight and body composition in type 2 diabetic men. DESIGN: Longitudinal, controlled clinical intervention study with exercise training consisting of an 8 week supervised program of aerobic exercise (75% VO(2) peak, 45 min), twice a week and intermittent exercise, once a week, on a bicycle ergometer. SUBJECTS: Sixteen men (age 45.4+/-7.2 y (mean+/-s.d.), HbA1c 8.15+/-1.7%, body mass index (BMI) 29.6+/-4.6 kg/m(2)) were randomly divided into two groups: trained group (n=8) and control group (n=8). MEASUREMENTS: Lipid, apo- and lipoprotein and DHEA concentrations. Cross-sectional areas of subcutaneous and visceral adipose tissue and mid-thigh muscle by magnetic resonance imaging. RESULTS: Training decreased visceral (153.25+/-38.55 vs 84.20+/-21.30 cm(2), P<0.001), subcutaneous (241.55+/-49.55 vs 198.00+/-39.99 cm(2), P<0.001) adipose tissue area and triglyceride levels (2.59+/-1.90 vs 1.79+/-1.08 nmol/l, P<0.05) and increased mid-thigh muscle cross-sectional area (148.30+/-36.10 vs 184.35+/-35.85 cm(2), P<0.001), and DHEA levels (11.00+/-3.10 vs 14.25+/-4.10 nmol/l, P<0.05) with no modification in body weight. Changes in triglycerides were negatively correlated with changes in DHEA (r=-0.81, P=0.03). This correlation was independent of changes in abdominal fat distribution. CONCLUSION: Training decreases abdominal fat depots, improves muscular mass and affects favourably triglyceride and DHEA levels. Changes in triglycerides and DHEA were inversely related.

Autosomal dominant type IIa hypercholesterolemia: evaluation of the respective contributions of LDLR and APOB gene defects as well as a third major group of defects.

Autosomal dominant type IIa hypercholesterolaemia (ADH) is characterised by an elevation of total plasma cholesterol associated with increased LDL particles. Numerous different molecular defects have been identified in the LDL receptor (LDLR) and few specific mutations in the apolipoprotein B (APOB) gene resulting in familial hypercholesterolaemia and familial defective apoB-100 respectively. To estimate the respective contribution of LDLR, APOB and other gene defects in this disease, we studied 33 well characterised French families diagnosed over at least three generations with ADH through the candidate gene approach. An estimation of the proportions performed with the HOMOG3R program showed that an LDLR gene defect was involved in approximately 50% of the families (P = 0.001). On the other hand, the estimated contribution of an APOB gene defect was only 15%. This low estimation of ADH due to an APOB gene defect is further strengthened by the existence of only two probands carrying the APOB (R3500Q) mutation in the sample. More importantly and surprisingly, 35% of the families in the sample were estimated to be linked to neither LDLR nor APOB genes. These data were confirmed by the exclusion of both genes through direct haplotyping in three families. Our results demonstrate that the relative contributions of LDLR and APOB gene defects to the disease are very different. Furthermore, our results also show that genetic heterogeneity is, generally, underestimated in ADH, and that at least three major groups of defects are involved. At this point, the contribution of the recently mapped FH3 gene to ADH cannot be assessed nor its importance in the group of 'non LDLR/non APOB' families.

Changes in plasma cystatin C after renal transplantation and acute rejection in adults.

BACKGROUND: Cystatin C has recently been proposed as an alternative marker of glomerular filtration rate. The diagnostic value of plasma cystatin C for the longitudinal assessment of kidney function after renal transplantation, however, has not been addressed. METHODS: Renal function was evaluated in 30 adults receiving renal transplants (46 +/- 9 years, mean +/- SD) and in 56 healthy controls (38 +/- 10 years) using cystatin C. Plasma cystatin C was determined daily starting the day of surgery and for 3 weeks after surgery by an immunonephelometric assay. RESULTS: Plasma concentration significantly decreased during the first week (-44% vs -29% for creatinine). Plasma cystatin C correlated with plasma creatinine (r = 0.741; P <0.0001) and the reciprocal of the creatinine clearance estimated by the Cockcroft-Gault formula (r = 0.882; P <0.001). In all three cases of acute renal impairment, the increase in plasma cystatin C values was more prominent than that of creatinine. CONCLUSIONS: Plasma cystatin C is an alternative and accurate marker of allograft function in adult transplant patients. Increased sensitivity compared with creatinine for the detection of acute reduction in glomerular filtration rate allows in some cases a more rapid diagnosis of acute rejection or treatment nephrotoxicity.

Sodium dodecyl sulfate-agarose gel electrophoresis of urinary proteins: application to multiple myeloma.

We evaluated a new sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) for urinary protein analysis in patients with multiple myeloma (MM; n = 47; ages, 62 +/- 2 years, mean +/- SE). Abnormal proteinuria (mean = 1872 +/- 360 mg/24 h) was present in 95% of the samples; 75% of the patients had some sign of renal dysfunction (glomerular and/or tubular) according to their SDS-AGE pattern. A band suggesting Bence Jones proteinuria (BJP) was detected in 40 vs 33 specimens by routine AGE. Immunofixation identified BJP in 38 patients; the calculated sensitivity of SDS-AGE for BJP was 97%. Excellent correlation (P <0.0001) was obtained with routine AGE (r = 0.994) and immunonephelometry (r = 0.963) for light chain quantification. SDS-AGE allows easy evaluation of renal dysfunction and shows high sensitivity for BJP detection. In a specialized laboratory, it is useful for following the progress of MM patients through the semiquantification of BJP.

Familial ligand-defective apolipoprotein B-100: simultaneous detection of the ARG3500-->GLN and ARG3531-->CYS mutations in a French population.

Familial ligand-defective apolipoprotein B-100 (FDB) is an autosomal dominant disorder leading to plasma LDL cholesterol elevation and coronary artery disease (CAD). Two specific mutations in the APOB gene--R3500Q and R3531C--induce FDB. We report an original method to detect both mutations simultaneously, based upon PCR-mediated, site-directed mutagenesis and double restriction of a unique PCR product. With this method we have investigated the prevalence of these mutations in 1,040 French patients. The R3500Q mutation was found in five probands. Genotypes were determined for 10 APOB polymorphic markers and were consistent with the common European ancestral haplotype previously reported. The only exception was one FDB proband who did not harbor the 48 repeat allele of the 3'HVR. Additionally, the first two R3531C mutations were identified in French probands. Genotypes were consistent with a previously reported haplotype, suggesting that this is another mutation of European ancestry.

Association of elevated lipoprotein(a) levels and coronary heart disease in NIDDM patients. Relationship with apolipoprotein(a) phenotypes.

Non-insulin-dependent diabetes mellitus (NIDDM) is a strong and independent risk factor for coronary heart disease. We assessed the potential relationship between plasma Lp(a) levels, apo(a) phenotypes and coronary heart disease in a population of NIDDM patients. Seventy-one patients with coronary heart disease, who previously have had transmural myocardial infarction, or significant stenosis on coronary angiography, or positive myocardial thallium scintigraphy, or in combination, were compared with 67 patients without coronary heart disease, who tested negatively upon either coronary angiography, myocardial thallium scintigraphy or a maximal exercise test. The prevalence of plasma Lp(a) levels elevated above the threshold for increased cardiovascular risk (> 0.30 g/l) was significantly higher (p = 0.005) in patients with coronary heart disease (33.8%) compared to the control group (13.4%). The relative risk (odds ratio) of coronary heart disease among patients with high Lp(a) concentrations was 3.1 (95% confidence interval, 1.31-7.34; p = 0.01). The overall frequency distribution of apo(a) phenotypes differed significantly between the two groups (p = 0.043). However, the frequency of apo(a) isoforms of low apparent molecular mass (< or = 700 kDa) was of borderline significance (p = 0.067) between patients with or without coronary heart disease (29.6% and 16.4%, respectively). In this Caucasian population of NIDDM patients, elevated Lp(a) levels were associated with coronary heart disease, an association which was partially accounted for by the higher frequency of apo(a) isoforms of small size. In multivariate analyses, elevated levels of Lp(a) were independently associated with coronary heart disease (odds ratio 3.48, p = 0.0233).

Lipoprotein(a) in diabetic patients and normoglycemic relatives in familial NIDDM.

OBJECTIVE: To compare lipoprotein(a) levels in diabetic patients and normoglycemic relatives in familial NIDDM and to assess whether Lp(a) is a risk factor for myocardial infarction in this population. RESEARCH DESIGN AND METHODS: We compared 577 patients and 261 normoglycemic relatives from 189 NIDDM multiplex families with 49 unrelated healthy individuals. Of the 577, 23 patients with previously documented myocardial infarction were further analyzed as a separate group. RESULTS: Lp(a) concentrations in diabetic patients, normoglycemic relatives, and the control group were not significantly different. Variance of Lp(a) in a given individual could not be accounted for by any clinical or biological parameter, but was strongly related to the mean Lp(a) value in his or her family. Diabetic patients with previous myocardial infarction (and their relatives) had significantly higher levels of Lp(a) than patients without coronary heart disease complaints. CONCLUSIONS: Lp(a) concentration in familial NIDDM was not related to the degree of glucose intolerance, but presented a strong familial aggregation. High Lp(a) levels seem to be an independent risk factor for myocardial infarction in this NIDDM cohort.

Efficacy and safety of simvastatin (alone or in association with cholestyramine). A 1-year study in 66 patients with type II hyperlipoproteinaemia.

The effects and safety of simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, were investigated alone or in association with cholestyramine in 66 patients with hypercholesterolaemia, in a 1-year study. In type IIa hypercholesterolaemia (41 patients), the association was more effective than simvastatin used alone in lowering total cholesterol (37% vs 29%) and LDL-cholesterol (45% vs 37%). In type IIb hypercholesterolaemia (23 patients), the association simvastatin-cholestyramine did not appear more effective than simvastatin used alone. The decrease of apoprotein B was parallel to the LDL-cholesterol decrease. Apoprotein A1 did not change significantly. The long-term safety of simvastatin was good. No lens opacity was noted. The most serious side-effect in our study was myolysis which occurred in two patients with a marked increase in creatine phosphokinase.

[Removal of low density lipoproteins on dextrans sulfate in 2 patients with familial monogenic hypercholesterolemia]. / Epuration des lipoprotéines de basse densité (LDL) sur sulfate de dextran chez deux patients affectés d'une hypercholestérolémie monogénique familiale.

Two patients-a 32 year old man with severe heterozygote familial hyperlipoproteinemia (FH) and a 9 years old girl with homozygote FH-were treated over eight months by LDL apheresis using dextran sulfate cellulose column (Liposorber, Kaneka, Japon). Plasma was separated from blood cells by filtration (TPE Cobe) or centrifugation (2,997 Cobe) through peripheral veins. An IV bolus of 10 IU/kg heparin was given together with local anti-coagulation with 55 g/l sodium citrate, 20 g/l citric acid at a ratio 1:25. Albumin supply was unnecessary. Plasma was removed every 2 weeks through liposorber LA 40 in the adult, and every week through liposorber LA 40 then 2 LA 15 in the child, mean plasma volume exchanged being 1.2 litres in the adult and 1.5 litres par session in the child. EFFECTIVENESS: the DSC column removed on the average 60 p. 100 of total cholesterol (TC) and 65 p. 100 of LDL.C. Apoproteins B levels were reduced by 58 p. 100. After each procedure there was a rapid increase in lipid levels to about the 80 to 90 p. 100 of pretreatment value. In the adult, we obtained levels of TC of less than 300 mg/dl with exchanges every 2 weeks combined with an HMG CoA reductase inhibitor (40 mg/day); in the child, with exchanges every week the same inhibitor did not permit a prolongation of the interval between 2 aphereses. SPECIFICITY: this was confirmed by elution of DSC column bound lipoproteins by 0.1 mol/l NaCl solution. However, the average removal of HDL.C and apoprotein A1 was respectively 31 p. 100 and 32 p. 100. Triglycerides levels were also reduced (48 p. 100). SAFETY: this was good in both cases. Using the filtration technic, hypotension was reported; this side effect did not appear with centrifugation. In the child, we observed immediate type reactions: nasal obstruction, headache and abdominal pain. The change in plasma protein concentration was caused by dilution and/or non specific absorption. CONCLUSION: LDL apheresis alone or combined with an HMG CoA reductase inhibitor is a safe technic, simple to manage without special equipment and producing marked LDL.C level reduction. However, there is also a reduction of HDL-C levels. Despite its high cost, it is a promising new approach to the treatment of FH.

Plasma lipids in juvenile chronic granulocytic leukaemia.

Plasma lipid parameters (triglycerides, total cholesterol and high density lipoprotein cholesterol) were measured in 7 children with juvenile chronic granulocytic leukaemia, of whom 3 were with and 4 without xanthomas. In all cases, whatever the stage of the disease, these parameters were extremely altered. Plasma triglycerides were generally increased, total and HDL cholesterols were very low. No relationship seems to exist between these values and the occurrence of xanthomas.

Specific modulation of surface receptors in J.774 macrophages by anchorage.

The J.774 murine macrophage cells were cultured in suspension in Teflon flasks. When allowed to attach on culture plastic dishes, a 2-3-fold increase in transferrin binding was observed. This occurred in 10 min, reached a steady state at 60 min, remained stable for several hours and was reversible after resuspension of the cells at 37 degrees C. The phenomenon was not dependent on the synthesis of new protein. An opposite change of acetyl LDL receptors was observed, with a threefold decrease of the binding 1 h after the attachment of the cells. The increase of transferrin binding affected almost equally the cell surface and the intracellular sites; therefore it could not be related to a simple shift between these two compartments. It is suggested that the attachment of the cells induced a recruitment of binding sites from a 'silent' pool of receptors. Serum factors, as well as phorbol esters and db-cAMP potentiated the effect of anchorage.

Identification of specific binding sites for acetylated low density lipoprotein in microvillous membranes from human placenta.

The ability of microvillous membranes isolated from human placenta to specifically bind human low density lipoprotein (LDL) modified by acetic anhydride has been investigated. The presence of saturable high affinity binding sites specific for [125I]acetyl-LDL was demonstrated. Scatchard analysis of the binding data, obtained at 4 degrees C, revealed a single class of sites with a mean KD value of 3.63 +/- 1.16 micrograms acetyl-LDL protein/ml, and a maximal binding capacity of 335.1 +/- 148.8 ng acetyl-LDL protein/mg of membrane protein. At 37 degrees C, the binding capacity was increased, while the KD value was not modified. The specificity of these binding sites was assessed by competition studies: unlabelled acetyl-LDL were effective competitors, whereas native LDL, VLDL and HDL3 were ineffective. Conversely, unlabelled acetyl-LDL failed to prevent the binding of native [125I]LDL to placental microvilli. The [125I]acetyl-LDL binding was partially inhibited (about 35%) by dextran sulfate and fucoidin, and was abolished by a pretreatment of the microvillous membranes with pronase. The binding sites specific for acetyl-LDL are present during all the gestation and are distinctly different from the binding sites for native LDL, previously characterized in placental microvilli. These 2 types of binding sites may be related to the high amount of cholesterol required by the human placenta for progesterone synthesis and trophoblastic growth.