WW-domain-containing oxidoreductase is associated with low plasma HDL-C levels.

Low serum HDL-cholesterol (HDL-C) is a major risk factor for coronary artery disease. We performed targeted genotyping of a 12.4 Mb linked region on 16q to test for association with low HDL-C by using a regional-tag SNP strategy. We identified one SNP, rs2548861, in the WW-domain-containing oxidoreductase (WWOX) gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent (p = 6.9 x 10(-7)). We extended our investigation to the population level by using two independent unascertained population-based Finnish cohorts, the cross-sectional METSIM cohort of 4,463 males and the prospective Young Finns cohort of 2,265 subjects. The combined analysis provided p = 4 x 10(-4) to 2 x 10(-5). Importantly, in the prospective cohort, we observed a significant longitudinal association of rs2548861 with HDL-C levels obtained at four different time points over 21 years (p = 0.003), and the T risk allele explained 1.5% of the variance in HDL-C levels. The rs2548861 resides in a highly conserved region in intron 8 of WWOX. Results from our in vitro reporter assay and electrophoretic mobility-shift assay demonstrate that this region functions as a cis-regulatory element whose associated rs2548861 SNP has a specific allelic effect and that the region forms an allele-specific DNA-nuclear-factor complex. In conclusion, analyses of 9,798 subjects show significant association between HDL-C and a WWOX variant with an allele-specific cis-regulatory function.

A nonsynonymous SNP within PCDH15 is associated with lipid traits in familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCHL) is a common lipid disorder characterized by the presence of multiple lipoprotein phenotypes that increase the risk of premature coronary heart disease. In a previous study, we identified an intragenic microsatellite marker within the protocadherin 15 (PCDH15) gene to be associated with high triglycerides (TGs) in Finnish dyslipidemic families. In this study we analyzed all four known nonsynonymous SNPs within PCDH15 in 1,268 individuals from Finnish and Dutch multigenerational families with FCHL. Association analyses of quantitative traits for SNPs were performed using the QTDT test. The nonsynonymous SNP rs10825269 resulted in a P = 0.0006 for the quantitative TG trait. Additional evidence for association was observed with the same SNP for apolipoprotein B levels (apo-B) (P = 0.0001) and total cholesterol (TC) levels (P = 0.001). None of the other three SNPs tested showed a significant association with any lipid-related trait. We investigated the expression of PCDH15 in different human tissues and observed that PCDH15 is expressed in several tissues including liver and pancreas. In addition, we measured the plasma lipid levels in mice with loss-of-function mutations in Pcdh15 (Pcdh15(av-Tg) and Pcdh15(av-3J)) to investigate possible abnormalities in their lipid profile. We observed a significant difference in plasma TG and TC concentrations for the Pcdh15(av-3J) carriers when compared with the wild type (P = 0.013 and P = 0.044, respectively). Our study suggests that PCDH15 is associated with lipid abnormalities.

Galanin preproprotein is associated with elevated plasma triglycerides.

OBJECTIVE: There is increasing physiological evidence in rodents connecting the neuropeptide galanin to triglyceride (TG) levels. We hypothesized that variation in the galanin preproprotein (GAL) gene may contribute to hypertriglyceridemia (HTG) in humans. METHODS AND RESULTS: We investigated GAL as a TG candidate gene by genotyping 4 tagSNPs in Dutch, Finnish, and Mexican familial combined hyperlipidemia (FCHL) families as well as in white combined hyperlipidemia cases/controls (n=2471). The common allele of rs2187331, residing in the promoter region of GAL, was significantly associated with HTG (probability value=0.00038). In an unascertained population sample of 4463 Finnish males, the rare allele of rs2187331 was associated with higher TGs (probability value=0.0028 to 0.00016). We also observed an allele specific difference with rs2187331 in reporter gene expression and nuclear factor binding in vitro. Furthermore, we detected differential expression of many key lipid genes in adipose tissue based on rs2187331 genotypes. CONCLUSIONS: The SNP rs2187331 is associated with HTG in FCHL and white combined hyperlipidemia cases/controls and influences TG levels in the population. Further studies are warranted to elucidate the allelic difference observed between FCHL and the general population. Functional evidence shows that rs2187331 has an allele specific cis-regulatory function and influences the expression of lipid related genes in adipose.

The ATF6-Met[67]Val substitution is associated with increased plasma cholesterol levels.

OBJECTIVE: Activating transcription factor 6 (ATF6) is a sensor of the endoplasmic reticulum stress response and regulates expression of several key lipogenic genes. We used a 2-stage design to investigate whether ATF6 polymorphisms are associated with lipids in subjects at increased risk for cardiovascular disease (CVD). METHODS AND RESULTS: In stage 1, 13 tag-SNPs were tested for association in Dutch samples ascertained for familial combined hyperlipidemia (FCHL) or increased risk for CVD (CVR). In stage 2, we further investigated the SNP with the strongest association from stage 1, a Methionine/Valine substitution at amino-acid 67, in Finnish FCHL families and in subjects with CVR from METSIM, a Finnish population-based cohort. The combined analysis of both stages reached region-wide significance (P=9 x 10(-4)), but this association was not seen in the entire METSIM cohort. Our functional analysis demonstrated that Valine at position 67 augments ATF6 protein and its targets Grp78 and Grp94 as well as increases luciferase expression through Grp78 promoter. CONCLUSIONS: A common nonsynonymous variant in ATF6 increases ATF6 protein levels and is associated with cholesterol levels in subjects at increased risk for CVD, but this association was not seen in a population-based cohort. Further replication is needed to confirm the role of this variant in lipids.

Association of stearoyl-CoA desaturase 1 activity with familial combined hyperlipidemia.

OBJECTIVE: Stearoyl-CoA desaturase 1 (SCD1) is the rate-limiting enzyme involved in the synthesis of monounsaturated fatty acids, and in mice SCD1 activity is associated with plasma triglyceride levels. We used the fatty acid desaturation index (the plasma ratio of 18:1/18:0) as a marker of SCD1 activity to investigate the relationship of SCD1 to familial combined hyperlipidemia (FCHL). METHODS AND RESULTS: The fatty acid desaturation index was measured in 400 individuals from 18 extended FCHL pedigrees. FCHL-affected individuals exhibited increased SCD1 activity when compared to unrelated controls (P < 0.0001). The fatty acid desaturation index was found to be highly heritable (h(2) = 0.48, P = 2.2 x 10(-11)) in this study sample. QTL analysis in 346 sibling pairs from 18 FCHL families revealed suggestive linkage of the desaturation index to chromosomes 3p26.1 to 3p13 (z = 2.7, P = 0.003), containing the peroxisome proliferator-activated receptor gamma (PPARgamma) gene, and 20p11.21 to 20q13.32 (z = 1.7, P = 0.04), containing the hepatocyte nuclear factor 4, alpha (HNF4alpha) gene. A specific haplotype of HNF4alpha was found to be associated with the desaturation index in these FCHL families (P = 0.002). CONCLUSIONS: Our results demonstrate that the fatty acid desaturation index is a highly heritable trait that is associated with the dyslipidemia observed in FCHL.

Upstream transcription factor 1 (USF1) in risk of type 2 diabetes: association study in 2000 Dutch Caucasians.

Type 2 diabetes shares substantial genetic and phenotypic overlap with familial combined hyperlipidemia. Upstream stimulatory factor 1 (USF1), a well-established susceptibility gene for familial combined hyperlipidemia, is postulated to be such a shared genetic determinant. We evaluated two established variants in familial combined hyperlipidemia (rs2073658 and rs3737787) for association with type 2 diabetes in two Dutch case-control samples (N=2011). The first case-control sample comprised 501 subjects with type 2 diabetes from the Breda cohort and 920 healthy blood bank donors of Dutch Caucasian origin. The second case-control sample included 211 subjects with type 2 diabetes, and 379 normoglycemic controls. SNP rs2073658 and SNP rs3737787 were in perfect linkage disequilibrium. In the first case-control sample, prevalence of the major allele was higher in patients than in controls (75% versus 71%, OR=1.25, p=0.018). A similar effect-size and -direction was observed in the second case-control sample (76% versus 72%, OR=1.22, p=0.16). A combined analysis strengthened the evidence for association (OR=1.23, p=0.006). Notably, the increased risk for type 2 diabetes could be ascribed to the major allele, and its high frequency translated to a substantial population attributable risk of 14.5%. In conclusion, the major allele of rs2073658 in the USF1 gene is associated with a modestly increased risk to develop type 2 diabetes in Dutch Caucasians, with considerable impact at the population level.

Effects of interacting networks of cardiovascular risk genes on the risk of type 2 diabetes mellitus (the CODAM study).

BACKGROUND: Genetic dissection of complex diseases requires innovative approaches for identification of disease-predisposing genes. A well-known example of a human complex disease with a strong genetic component is Type 2 Diabetes Mellitus (T2DM). METHODS: We genotyped normal-glucose-tolerant subjects (NGT; n = 54), subjects with an impaired glucose metabolism (IGM; n = 111) and T2DM (n = 142) subjects, in an assay (designed by Roche Molecular Systems) for detection of 68 polymorphisms in 36 cardiovascular risk genes. Using the single-locus logistic regression and the so-called haplotype entropy, we explored the possibility that (1) common pathways underlie development of T2DM and cardiovascular disease -which would imply enrichment of cardiovascular risk polymorphisms in "pre-diabetic" (IGM) and diabetic (T2DM) populations- and (2) that gene-gene interactions are relevant for the effects of risk polymorphisms. RESULTS: In single-locus analyses, we showed suggestive association with disturbed glucose metabolism (i.e. subjects who were either IGM or had T2DM), or with T2DM only. Moreover, in the haplotype entropy analysis, we identified a total of 14 pairs of polymorphisms (with a false discovery rate of 0.125) that may confer risk of disturbed glucose metabolism, or T2DM only, as members of interacting networks of genes. We substantiated gene-gene interactions by showing that these interacting networks can indeed identify potential "disease-predisposing allele-combinations". CONCLUSION: Gene-gene interactions of cardiovascular risk polymorphisms can be detected in prediabetes and T2DM, supporting the hypothesis that common pathways may underlie development of T2DM and cardiovascular disease. Thus, a specific set of risk polymorphisms, when simultaneously present, increases the risk of disease and hence is indeed relevant in the transfer of risk.

Parabolic relationship between plasma triacylglycerols and LDL-cholesterol in familial combined hyperlipidaemia: the multiple-type hyperlipidaemia explained?

FCHL (familial combined hyperlipidaemia) is a highly prevalent genetic lipid disorder that accounts for a substantial number of premature cardiovascular events. To date, FCHL has been complicated by the different lipid phenotypes that are present within one family and one individual patient over time. In the present study, we hypothesized that a parabolic relationship between plasma triacylglycerols (triglycerides) and LDL (low-density lipoprotein)-cholesterol can explain this so-called 'multiple-type hyperlipidaemia' in FCHL. Our hypothesis was tested in two well-documented FCHL cohorts [Maastricht (n=145) and Nijmegen (n=299)] that were followed over a 5-year interval. Three groups were constructed depending on plasma triacylglycerols: group A (individuals with both measurements below 1.5 mmol/l), group B (one measurement below and one measurement above 1.5 mmol/l) and group C (both measurement above 1.5 mmol/l). In both male, but not female, cohorts, a significant positive relationship between plasma triacylglycerols and LDL-cholesterol was observed in group A (P=0.02 for Maastricht cohort and P=0.001 for the Nijmegen cohort), a significant negative relationship in group C (P=0.01 for Maastricht cohort and P=0.02 for the Nijmegen cohort), and a relationship intermediate to group A and C in group B. In contrast, both apoB (apolipoprotein B) levels and the prevalence of cardiovascular disease were related with plasma triacylglycerols in a more linear fashion. In conclusion, a parabolic relationship between plasma triacylglycerols and LDL-cholesterol explains the 'multiple-type hyperlipidaemia' in FCHL. In addition, the linear relationship between triacylglycerols and both apoB levels and the prevalence of cardiovascular disease substantiate the use of apoB instead of LDL-cholesterol in the diagnosis of FCHL and the prediction of cardiovascular disease.

Plasma PAI-1 levels are independently related to fatty liver and hypertriglyceridemia in familial combined hyperlipidemia, involvement of apolipoprotein E.

BACKGROUND: Familial combined hyperlipidemia (FCHL) is a genetic form of dyslipidemia, which is characterized by an increased cardiovascular risk. The current study was conducted to investigate the relation of endothelial, inflammatory and fibrinolysis markers with the presence of hypertriglyceridemia and fatty liver in FCHL, in order to advance insight in their contribution to the cardiovascular risk profile. MATERIALS AND METHODS: Key plasma markers of low-grade inflammation, endothelial dysfunction and fibrinolysis were measured in 38 hypertriglyceridemic FCHL patients and 38 age and sex-matched spouses. The presence of fatty liver was determined with ultrasound. RESULTS: hsCRP, vWF, PAI-1, tPA and tPA/PAI-1 complex levels were significantly higher in hypertriglyceridemic FCHL patients compared to spouses (p<0.05). Subsequent analyses revealed that these increased levels were confined to FCHL patients with the fatty liver phenotype (n=25). Only PAI-1 and tPA levels were also elevated in the hypertriglyceridemic FCHL patients without fatty liver (n=13). Of interest, 11 hypertriglyceridemic non-FCHL patients with the E2/E2 genotype displayed significantly lower PAI-1 levels when compared to the overall FCHL population (p=0.001), implicating a role for apolipoprotein E in the relation of PAI-1 with plasma triglycerides. CONCLUSION: Markers of fibrinolysis were increased in all hypertriglyceridemic FCHL patients, whereas an increased state of endothelial dysfunction and inflammation was particularly observed in those hypertriglyceridemic FCHL patients who also have fatty liver. These results demonstrate the complex genesis of the unfavourable cardiovascular risk profile that is present in FCHL, and illustrate the potential risk of fatty liver above, and beyond hypertriglyceridemia per se.

USF1 contributes to high serum lipid levels in Dutch FCHL families and U.S. whites with coronary artery disease.

OBJECTIVE: Familial combined hyperlipidemia (FCHL) characterized by high serum total cholesterol and/or triglycerides (TGs) is a common dyslipidemia predisposing to coronary artery disease (CAD). Recently, the upstream transcription factor 1 (USF1) was linked and associated with FCHL and TGs in Finnish FCHL families. Here we examined the previously associated rs3737787 SNP in extended Dutch FCHL families (n=532) and in a cohort of US subjects who underwent diagnostic coronary angiography (n=1533). METHODS AND RESULTS: In males of the Dutch FCHL families, we observed significant sex-dependent associations between the common allele of rs3737787 and FCHL, TGs, and related metabolic traits (P=0.02 to 0.006). In the U.S. Whites, sex-dependent associations with TGs and related metabolic traits were observed for the common allele of rs3737787 in males (P=0.04 to 0.02) and rare allele in females (P=0.05 to 0.002). This intriguing relationship was further supported by the highly significant genotype x sex interactions observed for TGs in the Dutch and TGs and body mass index (BMI) in U.S. White subjects with CAD (P=0.0005 to 0.00004). CONCLUSIONS: These data show that USF1 influences several cardiovascular risk factors in a sex-dependent manner in Dutch FCHL families and U.S. Whites with CAD. A significant interaction between sex and genotype was shown to affect TGs and BMI.

Omega-3 carboxylic acids in patients with severe hypertriglyceridemia: EVOLVE II, a randomized, placebo-controlled trial.

BACKGROUND: Adult patients with severe hypertriglyceridemia (SHTG) are at increased risk of developing acute pancreatitis and cardiovascular disease. Omega-3 carboxylic acids (OM3-CA) are approved for treatment as an adjunct to diet to reduce triglyceride (TG) concentrations in patients with SHTG. OBJECTIVE: The aim of the study was to assess efficacy and safety of the intermediate dose of OM3-CA (2 g daily), compared with olive oil 2 g daily, in reducing serum TG and lipid concentrations in patients with SHTG. METHODS: A randomized, double-blind, olive oil-controlled, parallel-group trial involving 162 adults with qualifying serum TG concentrations of at least 500 mg/dL (5.65 mmol/L) and <2500 mg/dL (28.25 mmol/L; <2000 mg/dL [22.60 mmol/L] in Canada). The treatment period after randomization was 12 weeks. Blood samples for measurement of fasting serum lipid concentrations were taken at baseline, 6, 10, and 12 weeks. RESULTS: Treatment with OM3-CA 2 g daily led to a significant reduction in TG concentrations (median of differences, -14.2% [95% confidence interval: -26.2%, -2.8%; P = .017]) and non-high-density lipoprotein cholesterol concentrations (median of differences, -9.0% [95% confidence interval: -14.8%, -2.8%; adjusted P = .018]) from baseline to the Week 12 endpoint, when compared with olive oil 2 g daily. These treatment effects were more pronounced in patients with qualifying TG concentrations >885 mg/dL (10 mmol/L). CONCLUSION: An intermediate dose of OM3-CA (2 g daily) significantly lowers TG and non-high-density lipoprotein cholesterol concentrations in patients with SHTG and may benefit individuals at risk of acute pancreatitis and cardiovascular disease.

Activating transcription factor 6 polymorphisms and haplotypes are associated with impaired glucose homeostasis and type 2 diabetes in Dutch Caucasians.

CONTEXT: Activating transcription factor 6 (ATF6) is critical for initiation and full activation of the unfolded protein response. An association between genetic variation in ATF6 and type 2 diabetes (DM2) was recently reported in Pima Indians. OBJECTIVES: To investigate the broader significance of this association for DM2, replication studies in distinct ethic populations are required. We investigated ATF6 for its association with DM2 in Dutch Caucasians. DESIGN/SETTING: A genetic association study was conducted at an academic research laboratory. STUDY PARTICIPANTS: Two independent Dutch cohorts were studied. Cohort 1 (n = 154) was used to evaluate genetic variation in the ATF6 gene in relation to glucose homeostasis in the general population. Cohort 2 (n = 798) consisted of patients with DM2, impaired glucose tolerance, impaired fasting glucose, and normoglycemic control subjects, and was used to investigate ATF6 polymorphisms for their contribution to disturbed glucose homeostasis and DM2. MAIN OUTCOME MEASURES: There were 16 tag single nucleotide polymorphisms genotyped in all subjects of both cohorts. Those single nucleotide polymorphisms included three nonsynonymous coding variants and captured all common allelic variation of ATF6. RESULTS: Our data show that common ATF6 variants are associated with elevated glucose levels in the general population (cohort 1, P = 0.005-0.05). Furthermore, the majority of these variants, and haplotypes thereof, were significantly associated with impaired fasting glucose, impaired glucose tolerance, and DM2 (cohort 2, P = 0.006-0.05). Associated variants differ from those identified in Pima Indians. CONCLUSIONS: Our results strengthen the evidence that one or more variants in ATF6 are associated with disturbed glucose homeostasis and DM2.

Fatty liver is an integral feature of familial combined hyperlipidaemia: relationship with fat distribution and plasma lipids.

Overproduction of VLDL (very-low-density lipoprotein) particles is an important cause of FCHL (familial combined hyperlipidaemia). It has been shown recently that VLDL production is driven by the amount of hepatic fat. The present study was conducted to determine the prevalence of fatty liver in relation to the different fat compartments and lipid parameters in FCHL. A total of 68 FCHL patients, 110 normolipidaemic relatives and 66 spouses underwent ultrasound of the abdominal region to estimate the amount of subcutaneous, visceral and hepatic fat. Skinfold callipers were used to measure subcutaneous fat of the biceps, triceps, subscapular and supra-iliacal regions. Fatty liver was observed in 18% of the spouses, 25% of the normolipidaemic relatives and 49% of the FCHL patients. After adjustment for age, gender and body mass index, the prevalence of fatty liver was significantly higher in FCHL patients compared with spouses [OR (odds ratio), 3.1; P=0.03], and also in the normolipidaemic relatives compared with spouses (OR, 4.0; P=0.02), whereas no differences were observed between FCHL patients and normolipidaemic relatives (OR, 0.8; P=0.58). In the normolipidaemic relatives and FCHL patients combined, both visceral fat mass and subcutaneous abdominal fat were independent predictors of fatty liver (P<0.001 for both fat compartments; FCHL status corrected). Of interest, fatty liver stages were correlated with both VLDL-apoB (apolipoprotein B) and VLDL-triacylglycerols (triglycerides) in a representative subset (n=69) of patients and relatives (r(2)=0.12, P=0.006; and r(2)=0.18, P=0.001 respectively). These results show that fatty liver is a central aspect of FCHL, i.e. patients and normolipidaemic relatives. Both visceral and subcutaneous adiposity contribute to its 3-4-fold higher risk in FCHL.

Five-year follow-up of waist circumference, insulin and ALT levels in familial combined hyperlipidaemia.

INTRODUCTION: Familial combined hyperlipidemia (FCHL), an entity with many features of the metabolic syndrome, is characterized by changes in cholesterol and triglyceride phenotype over time. This study was conducted to investigate the relation of alanineaminotransferase (ALT) levels, used as a surrogate for the amount of hepatic fat, with the switch in triglyceride phenotype and the increased susceptibility to develop hypertriglyceridemia in FCHL. METHODS: Body mass index, waist circumference, plasma triglycerides, insulin and ALT levels were measured in 145 FCHL family members and 54 spouses at baseline and after a five-year follow-up. RESULTS: A switch from normotriglyceridemia to hypertriglyceridemia or vice versa, as observed in 22 of 145 FCHL family members, was associated with changes in plasma ALT levels (p=0.001), but not with insulin levels or waist circumference. In five-year follow-up, an intra-individual relation was observed between waist circumference and both plasma triglycerides, insulin and ALT levels. For each waist circumference FCHLpatients, but not their normolipidemic relatives, exhibited higher triglyceride and insulin levels than spouses (p<0.001). Remarkably, both FCHL patients and the normolipidemic relatives showed higher ALT levels for each waist circumference as compared to spouses(p<0.001 for FCHL patients, p=0.035 for normolipidemic relatives). CONCLUSION: The present study shows that the longitudinal relation abdominal obesity-ALT is more specific for all FCHL family members, i.e. patients and their normolipidemic relatives, than the relation abdominal obesity-triglycerides. Additionally,the association of ALT with the switch in triglyceride phenotype suggests a central role of the liver in the pathogenesis of FCHL.

Fatty liver--based identification of two distinct hypertriglyceridemic subgroups in familial combined hyperlipidemia.

The present study was conducted to investigate whether the fatty liver phenotype could be helpful in the identification of subgroups with distinct metabolic properties and lipid profiles within familial combined hyperlipidemia (FCHL). One hundred eighty-five FCHL family members participated in the current study; 38 subjects were found to be hypertriglyceridemic, of whom 66% showed evidence of fatty liver as measured with ultrasound. A detailed comparison between the hypertriglyceridemic FCHL subjects with (n = 25) and without (n = 13) fatty liver revealed that, despite very similar plasma triglyceride levels (3.5 vs 3.2 mmol/L in subjects with and without fatty liver, respectively), the fatty liver subgroup presented with significantly higher body mass index, visceral adipose tissue (ultrasound), insulin, and alanine aminotransferase levels. Moreover, very low-density lipoprotein (VLDL) subclass analysis showed that the VLDL2 fraction of the fatty liver subgroup contained significantly less cholesterol and triglycerides (P = .02 for both parameters), which was likely explained by a decreased VLDL2 particle number because VLDL2 apolipoprotein B levels tended to be lower (P = .08). These data indicate that hypertriglyceridemic FCHL subjects may belong to metabolically distinct subgroups and suggest that a refinement of the hypertriglyceridemic FCHL phenotype by adding information on fatty liver will eventually facilitate the elucidation of its complex genetic background.

Up-regulation of CD36/FAT in preadipocytes in familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCHL) shows many features of the metabolic syndrome. The strong genetic component makes it an excellent model to study the genetic background of metabolic syndrome and insulin resistance. Adipose tissue is believed to contribute to, or even underlie, the FCHL phenotype and is an interesting target tissue for gene expression studies. However, interpretation of adipose tissue gene expression experiments is complex since expression differences cannot only arise as a direct consequence of a genetic trait, but may also reflect an adaptation to metabolic influences at the cellular level. In the present study, we measured gene expression levels in cultured primary human preadipocytes from FCHL and control subjects. Since isolated preadipocytes were allowed to replicate for weeks under standardized conditions, the contribution of previous metabolic influences is rather small whereas genetic defects are preserved and expressed in vitro. The main finding was up-regulation of CD36/FAT in FCHL preadipocytes, confirmed in two independent groups of subjects, and a concomitant increase in CD36/FAT-mediated fatty acid uptake. CD36/FAT overexpression has previously been shown to be associated with other insulin-resistant states. The present data suggest that CD36/FAT overexpression in FCHL occurs very early in adipocyte differentiation and may be of genetic origin.

Biochemical and genetic association of plasma apolipoprotein A-II levels with familial combined hyperlipidemia.

Apolipoprotein A-II (apoA-II) is a major protein on high-density lipoprotein (HDL) particles, and in mice, its levels are associated with triglyceride and glucose metabolism. In particular, transgenic mice overexpressing apoA-II exhibit hypertriglyceridemia, increased body fat, and insulin resistance, whereas apoA-II-null mice have decreased triglycerides and increased insulin sensitivity. Given the phenotypic overlap between familial combined hyperlipidemia (FCH) and apoA-II transgenic mice, we investigated the relationship of apoA-II to this disorder. Despite having lower HDL-cholesterol (HDL-C), FCH subjects had higher apoA-II levels compared with unaffected relatives (P<0.00016). Triglyceride and HDL-C levels were significant predictors of apoA-II, demonstrating that apoA-II variation is associated with several FCH-related traits. After adjustment for multiple covariates, there was evidence for the heritability of apoA-II levels (h2=0.15; P<0.02) in this sample. A genome scan for apoA-II levels identified significant evidence (LOD=3.1) for linkage to a locus on chromosome 1q41, coincident with a suggestive linkage for triglycerides (LOD score=1.4). Thus, this locus may have pleiotropic effects on apoA-II and FCH traits. Our results demonstrate that apoA-II is biochemically and genetically associated with FCH and may serve as a useful marker for understanding the mechanism by which FCH develops.

Locus for elevated apolipoprotein B levels on chromosome 1p31 in families with familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCH), a common cause of premature coronary artery disease, is genetically complex and poorly understood. Recently, a major locus on chromosome 1q21-23 exhibiting highly significant linkage was identified in Finnish FCH families by use of a parametric analysis. We now report highly significant evidence of linkage (maximum LOD score 3.8, recombination fraction 0) of an important FCH phenotype, elevated apolipoprotein B (apoB) levels, to a distinctly separate locus on chromosome 1p31 in Dutch pedigrees. ApoB is the major protein on very low density and low density lipoproteins, and elevated apoB levels have been used as a surrogate trait for FCH. Additional microsatellite markers in the 1p31 region were genotyped, and evidence of linkage improved (maximum LOD score 4.7) in a multipoint analysis of two markers in the peak region. The leptin receptor gene resides within this locus and is involved in obesity and insulin/glucose homeostasis. However, there was no evidence of an association between leptin receptor and apoB levels, raising the possibility that another gene on this chromosomal region contributes to elevated apoB levels in this Dutch population. This is one of the first loci identified for apoB levels in humans and is the second major locus implicated in the genetic etiology of FCH.

Association of the APOLIPOPROTEIN A1/C3/A4/A5 gene cluster with triglyceride levels and LDL particle size in familial combined hyperlipidemia.

The APOLIPOPROTEIN (APO)A1/C3/A4/A5 gene cluster on chromosome 11 has been hypothesized to be a modifier of plasma triglycerides in FCH. In the present study, we extended previous association analyses of the gene cluster to include APOA5, a newly discovered member of the cluster. Eight SNPs across the APOA1/C3/A4/A5 gene region were analyzed in 78 FCH probands and their normolipidemic spouses as well as in 27 Dutch FCH families. Of the individual SNPs tested in the case-control panel, the strongest evidence of association was obtained with SNPs in APOA1 (P=0.001) and APOA5 (P=0.001). A single haplotype defined by a missense mutation in APOA5 was enriched 3-fold in FCH probands when compared with the normolipidemic spouses (P=0.001) and a second haplotype was significantly enriched in the spouses (P=0.001). Family-based tests also indicated significant association of triglyceride levels and LDL particle size with the investigated SNPs of APOC3 and APOA5. These findings suggest that genetic variation in the APOA1/C3/A4/A5 gene cluster acts as a modifier of plasma triglyceride levels and LDL particle size within FCH families and furthermore indicate that a number of haplotypes may contribute to FCH.

Cardiovascular safety assessment of pramlintide in type 2 diabetes: results from a pooled analysis of five clinical trials.

BACKGROUND: This report evaluated the cardiovascular safety of the amylin analog pramlintide-an existing diabetes injectable treatment-by comparing relevant cardiovascular adverse events (AEs) reported in previous phase 3 and 4 clinical trials among patients receiving pramlintide and those receiving control treatments. METHODS: Cardiovascular safety of pramlintide was assessed using accepted regulatory medical definitions of AEs reported in five randomized, controlled phase 3 and 4 trials of 16-52 weeks' duration in adults with type 2 diabetes. The original trials compared pramlintide (90-120 mcg twice daily or 30-150 mcg three times daily) with placebo (four studies) or a mealtime rapid-acting insulin analog (one study). Background therapies included insulin alone or in combination with oral glucose-lowering agents. AE data obtained from clinical study reports were combined into one database and analyzed for the intention-to-treat population of 2016 patients (pramlintide, n = 1434; pooled comparator, n = 582). The primary analysis compared reported major adverse cardiovascular events (MACE) between pramlintide and control. RESULTS: The incidence of reported MACE was similar between pramlintide (4.7 %) and pooled comparators (4.5 %). Secondary analyses included MACE relative risk and hazard ratio point estimates, which ranged from 0.86 to 0.93 for pramlintide relative to comparator treatment; the upper limit of the two-sided 95 % confidence interval did not exceed the threshold of 1.8. CONCLUSIONS: Both the point estimate of the reported MACE frequency and estimated risk ratios showed that mealtime pramlintide as an adjunct to insulin conferred no increased risk of cardiovascular AEs in patients with type 2 diabetes using insulin.