Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.

Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) levels. Here we have identified two Tangier disease (TD) families, confirmed 9q31 linkage and refined the disease locus to a limited genomic region containing the gene encoding the ATP-binding cassette transporter (ABC1). Familial HDL deficiency (FHA) is a more frequent cause of low HDL levels. On the basis of independent linkage and meiotic recombinants, we localized the FHA locus to the same genomic region as the TD locus. Mutations in ABC1 were detected in both TD and FHA, indicating that TD and FHA are allelic. This indicates that the protein encoded by ABC1 is a key gatekeeper influencing intracellular cholesterol transport, hence we have named it cholesterol efflux regulatory protein (CERP).

Age and residual cholesterol efflux affect HDL cholesterol levels and coronary artery disease in ABCA1 heterozygotes.

We and others have recently identified mutations in the ABCA1 gene as the underlying cause of Tangier disease (TD) and of a dominantly inherited form of familial hypoalphalipoproteinemia (FHA) associated with reduced cholesterol efflux. We have now identified 13 ABCA1 mutations in 11 families (five TD, six FHA) and have examined the phenotypes of 77 individuals heterozygous for mutations in the ABCA1 gene. ABCA1 heterozygotes have decreased HDL cholesterol (HDL-C) and increased triglycerides. Age is an important modifier of the phenotype in heterozygotes, with a higher proportion of heterozygotes aged 30-70 years having HDL-C greater than the fifth percentile for age and sex compared with carriers less than 30 years of age. Levels of cholesterol efflux are highly correlated with HDL-C levels, accounting for 82% of its variation. Each 8% change in ABCA1-mediated efflux is predicted to be associated with a 0.1 mmol/l change in HDL-C. ABCA1 heterozygotes display a greater than threefold increase in the frequency of coronary artery disease (CAD), with earlier onset than unaffected family members. CAD is more frequent in those heterozygotes with lower cholesterol efflux values. These data provide direct evidence that impairment of cholesterol efflux and consequently reverse cholesterol transport is associated with reduced plasma HDL-C levels and increased risk of CAD.

Common sequence variants of lipoprotein lipase: standardized studies of in vitro expression and catalytic function.

We have assessed the functional activity of three common sequence variants of human lipoprotein lipase (LPL). Two of these, Asn291Ser and Asp9Asn arise from missense mutations while the third, Ser447Ter, derives from a nonsense mutation, truncating LPL by two residues. As previous in vitro studies have produced conflicting results, we have re-analyzed the catalytic function of these variants using the COS cell transfection system, under optimized and standardized experimental protocols. We found the Asn291Ser variant to manifest with a decrease in catalytic activity (57% of normal) due to a reduction in secretion and stability of the active homodimeric form. The Asp9Asn variant also showed a significant decrease in catalytic activity (85% of normal), but this was found to be due to a decreased rate of secretion only, as the homodimeric form was stable. The findings for these mutants contrasted with those of the Ser447Ter truncation variant which proved to be catalytically normal; this variant also manifested normal homodimer stability. The truncated variant did however, present with a higher total secreted mass level (131%) than control LPL. This was most likely due to enhanced secretion of the monomeric form. None of these mutations exhibited defects in binding affinity to cell surface proteoglycans. Each of these variants deviated significantly from normal as regards to their secreted activity or mass levels in the COS cell transfection system.

Common genetic variation in ABCA1 is associated with altered lipoprotein levels and a modified risk for coronary artery disease.

BACKGROUND: Low plasma HDL cholesterol (HDL-C) is associated with an increased risk of coronary artery disease (CAD). We recently identified the ATP-binding cassette transporter 1 (ABCA1) as the major gene underlying the HDL deficiency associated with reduced cholesterol efflux. Mutations within the ABCA1 gene are associated with decreased HDL-C, increased triglycerides, and an increased risk of CAD. However, the extent to which common variation within this gene influences plasma lipid levels and CAD in the general population is unknown. METHODS AND RESULTS: We examined the phenotypic effects of single nucleotide polymorphisms in the coding region of ABCA1. The R219K variant has a carrier frequency of 46% in Europeans. Carriers have a reduced severity of CAD, decreased focal (minimum obstruction diameter 1.81+/-0.35 versus 1.73+/-0.35 mm in noncarriers, P:=0.001) and diffuse atherosclerosis (mean segment diameter 2.77+/-0.37 versus 2.70+/-0.37 mm, P:=0.005), and fewer coronary events (50% versus 59%, P:=0.02). Atherosclerosis progresses more slowly in carriers of R219K than in noncarriers. Carriers have decreased triglyceride levels (1.42+/-0.49 versus 1.84+/-0.77 mmol/L, P:=0.001) and a trend toward increased HDL-C (0.91+/-0.22 versus 0.88+/-0.20 mmol/L, P:=0.12). Other single nucleotide polymorphisms in the coding region had milder effects on plasma lipids and atherosclerosis. CONCLUSIONS: These data suggest that common variation in ABCA1 significantly influences plasma lipid levels and the severity of CAD.

Common mutations in the lipoprotein lipase gene (LPL): effects on HDL-cholesterol levels in a Chinese Canadian population.

BACKGROUND: favorable lipid profiles including low total serum cholesterol (TC), TC/HDL-cholesterol (HDL-C) ratio and elevated HDL-C levels have been previously reported in Chinese living in China. More recent data, however, suggests a changing trend toward decreased HDL-C and increased TC and LDL cholesterol (LDL-C) in Chinese populations. Environmental factors likely contribute, in part, to these findings. However, genetic factors contributing to lipoprotein metabolism may also play a role in determining the lipid/lipoprotein phenotype observed in Chinese populations. Lipoprotein lipase (LPL) mutations have been associated with altered HDL-C concentrations in Caucasians but have not yet been studied in a large population of Chinese descent. METHODS: 1577 Chinese Canadians of Cantonese descent were recruited for a cardiovascular risk factor study. The frequency and effect of three LPL gene polymorphisms [Asp9Asn (D9N, n=374), Asn291Ser (N291S, n=321) and Ser447-Ter (S447X, n=403)] on serum HDL-C concentrations was assessed. All the three polymorphisms have been shown to alter HDL-C levels in different Caucasian populations. RESULTS: lower TC, LDL-C, and TG and higher HDL-C were observed in both male and female Chinese Canadian subjects compared to other population samples. The D9N and N291S LPL polymorphisms were identified in 1/374 (0.3%) and 5/321 (1.6%) subjects, respectively. Carrier frequency of the S447X mutation was (102/403) 25.3%. This S447X polymorphism was observed with higher frequency in males with HDL-C levels in the highest tertile compared with those in the lowest HDL-C tertile (carrier frequencies 37.3 vs. 19.4%) (P=0.046). CONCLUSION: in this cohort of Chinese Canadians, the serum lipid profiles were more favorable than what has been reported for Caucasian Canadians. A favorable spectrum of polymorphisms in the LPL gene may mitigate the adverse effects of western lifestyle on plasma lipoproteins in this cohort of Cantonese Canadians.

Maternal expression of functional lipoprotein lipase and effects on body fat mass and body condition scores of mature cats with lipoprotein lipase deficiency.

OBJECTIVE: To assess effects of deficiency of lipoprotein lipase (LPL) on body condition scores and lean and fat body masses of adult cats. ANIMALS: 12 cats without LPL mutations and 23 cats that were heterozygous or homozygous carriers of the Gly412Arg LPL mutation. PROCEDURE: Lean and fat body masses were estimated by use of body condition scores and change in enrichment of serum after IV administration of deuterium oxide. Mass spectroscopy and infrared absorbance methods were used to determine deuterium enrichment. RESULTS: Fat body mass (mean +/- SD; 0.2 +/- 0.1 kg) and percentage body fat (6.2 +/- 1.4%) of homozygotes were significantly less than those of clinically normal cats and heterozygotes (0.7 +/- 0.1 kg, 18.2 +/- 1.6% and 0.5 +/- 0.1 kg, 15.6 +/- 1.7%, respectively). Homozygous offspring of homozygous dams had significantly less fat body mass (0.1 +/- 0.1 kg) and percentage body fat (2.1 +/- 1.0%) than homozygous offspring of heterozygous dams (0.3 +/- 0.1 kg and 9.2 +/- 1.7%, respectively). Lean body mass did not differ significantly among groups. For all groups, percentage body fat was significantly correlated with body condition score (r= 0.65), and body condition scores supported findings for fat body mass. CONCLUSIONS AND CLINICAL RELEVANCE: Deficiency of LPL activity in cats diminishes stores of body fat. This is consistent with a low rate of de novo synthesis of fat. The effect of dam on body masses in mature LPL-deficient cats indicates nutrient programming of adipose formation during gestation or lactation.

Effect of insulin on excitatory synaptic transmission onto dopamine neurons of the ventral tegmental area in a mouse model of hyperinsulinemia.

Obesity has drastically increased over the last few decades. Obesity is associated with elevated insulin levels, which can gain access to the brain, including into dopamine neurons of the ventral tegmental area (VTA), a brain region critical for mediating reward-seeking behavior. Synaptic plasticity of VTA dopamine neurons is associated with altered motivation to obtain reinforcing substances such as food and drugs of abuse. Under physiological circumstances, insulin in the VTA can suppress excitatory synaptic transmission onto VTA dopamine neurons and reduce aspects of palatable feeding behavior. However, it is unknown how insulin modulates excitatory synaptic transmission in pathological circumstances such as hyperinsulinemia. Using patch-clamp electrophysiology, we demonstrate that, in a hyperinsulinemic mouse model, insulin has reduced capacity to cause a synaptic depression of VTA dopamine neurons, although both low-frequency stimulation-induced long-term depression and cannabinoid-induced depression were normal. These results suggest that insulin action in the VTA during pathological hyperinsulinemia is disrupted and may lead to increased feeding behavior.

The LPL S447X cSNP is associated with decreased blood pressure and plasma triglycerides, and reduced risk of coronary artery disease.

Linkage of the lipoprotein lipase (LPL) gene to blood pressure levels has been reported. The LPL S447X single nucleotide polymorphism (cSNP) has been associated with decreased triglycerides (TG), increased high density lipoprotein cholesterol, and a decreased risk of coronary artery disease (CAD), which may occur independently of its beneficial lipid changes. To investigate the relationship between LPL S447X cSNP and these parameters, we studied a cohort of individuals with familial hypercholesterolemia in whom blood pressures and information regarding the use of blood pressure lowering medications were available. Carriers of the S447X variant had decreased TG (1.21+/-0.47 vs. 1.52+/-0.67, p<0.001) and a trend towards decreased vascular disease (12.7 vs. 19.5%) compared to non-carriers. More interestingly, however, carriers of this cSNP had decreased diastolic blood pressure compared to non-carriers (78+/-10 vs. 82+/-11, p=0.002), evident in both men and women, youths and adults, with similar trends for systolic blood pressure. Furthermore, the decrease in blood pressure appeared independent of the decrease in TG (p=0.02), suggesting that the LPL protein may have a direct influence on the vascular wall. This suggests an additional mechanism whereby this variant may have protective effects, independent of changes in plasma lipid levels.

Differences in the phenotype between children with familial defective apolipoprotein B-100 and familial hypercholesterolemia.

Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited genetic disorder resulting from a point mutation in the apolipoprotein (apo) B gene and is associated with significantly elevated plasma total and LDL cholesterol levels. Despite numerous descriptions outlining the phenotype of children with familial hypercholesterolemia (FH), no study has described the biochemical and clinical phenotype in a cohort of children with FDB. The phenotypes of FH and FDB, therefore, have not been compared in children. We have studied a cohort of 38 Dutch children (all <20 years old) with FDB from 21 different families. Lipid and lipoprotein levels and the clinical phenotype were compared with 97 age-matched FH heterozygotes, as defined by molecular analysis, and with age-matched non-FDB, non-FH control subjects. Female FDB carriers (n=23) had significantly lower total cholesterol (P<.001), LDL cholesterol (P=.001), total cholesterol:HDL ratio (P<.001), and apoB levels (P=.001) than age-matched female FH heterozygotes (n=50). Similar results were noted in male FDB carriers (n=15) compared with male FH heterozygotes (n=47; P=.005, P=.007, P=.014, and P=.074, respectively). Within the FDB group, female FDB heterozygotes had higher LDL cholesterol (P=.038) and a trend to higher total cholesterol levels (P=.165) than age-matched males. Both male and female FDB carriers had significantly higher total cholesterol, LDL cholesterol, and total cholesterol:HDL ratio than age- and sex-matched control subjects, which was evident even in children <10 years of age, providing additional evidence that this mutation is penetrant in early life. These results provide evidence for a milder biochemical phenotype in children with FDB than in children with FH. The phenotype observed is intermediate between that of control subjects and FH heterozygotes matched for age and sex. As the incidence of coronary artery disease is related to both the extent and duration of cholesterol elevation, our findings might explain in part the lower incidence of clinical atherosclerosis seen in adults with this condition than in adults with FH.

Cholesterol efflux regulatory protein, Tangier disease and familial high-density lipoprotein deficiency.

Cellular cholesterol efflux, by which cholesterol is transported from peripheral cells to HDL acceptor molecules for transport to the liver, is the first step of reverse cholesterol transport. Two genetic disorders, Tangier disease and some cases of familial HDL deficiency, have defects of cellular cholesterol efflux. The recent discovery of mutations in the ABC1 gene, which encodes the cholesterol efflux regulatory protein, in both these disorders establishes cholesterol efflux regulatory protein as a rate-limiting factor in reverse cholesterol transport.

Relationship between lipoprotein lipase and high density lipoprotein cholesterol in mice: modulation by cholesteryl ester transfer protein and dietary status.

Plasma lipoprotein lipase (LPL) activity correlates with high density lipoprotein (HDL) cholesterol levels in humans. However, in several mouse models created either through transgenesis or targeted inactivation of LPL, no significant changes in HDL cholesterol values have been evident. One possible explanation for this species difference could be the absence of plasma cholesteryl ester transfer protein (CETP) activity in mice. To explore this possibility and further investigate interactions between LPL and CETP modulating HDL cholesterol levels in vivo, we examined the relationship between LPL activity and HDL levels in mice expressing the simian CETP transgene, compared with littermates not carrying the CETP gene. On a chow diet, increasing LPL activity was associated with a trend towards increased HDL levels (51 +/- 29 vs. 31 +/- 4 mg/dL highest vs. lowest tertiles of LPL activity, P = 0.07) in mice expressing CETP, while no such effects were seen in the absence of CETP (65 +/- 12 vs. 61 +/- 15 mg/ dL). Furthermore, in the presence of CETP, a significant positive correlation between LPL activity and HDL cholesterol was evident (r = 0.15, P = 0.006), while in the absence of CETP no such correlation was detected (r = 0.15, P = 0.36), highlighting the interactions between LPL and CETP in vivo. When mice were challenged with a high fat, high carbohydrate diet, strong correlations between LPL activity and HDL cholesterol were seen in both the presence (r = 0.45, P = 0.03) and absence (r = 0.73, P < 0.001) of CETP. Therefore, under altered metabolic contexts, such as those induced by dietary challenge, the relation between LPL activity and HDL cholesterol may also become evident. Here we have shown that both genetic and environmental factors may modulate the association between LPL activity and HDL cholesterol, and provide explanations for the absence of any changes in HDL values in mice either transgenic or with targeted disruption of the LPL gene.

A frequently occurring mutation in the lipoprotein lipase gene (Asn291Ser) results in altered postprandial chylomicron triglyceride and retinyl palmitate response in normolipidemic carriers.

An Asn291Ser mutation in exon 6 of the lipoprotein lipase gene (LPL) frequently occurs in Caucasians (2-4%) and results in a partial catalytic defect. Although this mutation may be associated with low HDL cholesterol and elevated triglyceride levels, some carriers are normolipidemic and may have LPL activity in the normal range in the fasting state. To assess in vivo the influence of dietary stress on the function of this mutation, we have performed oral fat load studies on three unrelated normolipidemic Asn291Ser carriers and compared these results to five healthy controls and to a subject with a clear 50% reduction in LPL activity compared with controls. The Asn291Ser carriers exhibited a more pronounced postprandial response compared with non-carriers as evidenced by higher chylomicron triglyceride (TG) and chylomicron retinyl palmitate peaks (P = 0.03 and P = 0.02, respectively). Significantly higher area under response curves were also seen for both chylomicron triglycerides (P = 0.02) and chylomicron retinyl palmitate (P = 0.01) when compared with non-carriers. These results provide further in vivo evidence for the functional effects of this common mutation despite normal fasting lipid levels. These data suggest that even though subjects with this mutation may be normolipidemic in the fasting state, environmental stress such as an oral fat load may unmask the catalytic defect and result in significant disturbances in postprandial chylomicron metabolism.

Diet-induced atherosclerosis in the domestic cat.

The domestic cat has not been used in studies of atherosclerosis, with the exception of a single study published in 1970. We have further evaluated the susceptibility of the domestic cat to diet-induced atherosclerosis, the ultimate intent being to discern the atherogenic risk due to lipoprotein lipase deficiency in an affected feline kindred with a phenotype very similar to that of the human form of this condition. We subjected a group of normal domestic cats to a moderately high-fat, cholesterol-enriched diet (30% fat and 3% cholesterol) for a period of 2 to 8 months. Plasma lipid levels were monitored monthly. At the time of killing, all organs and the entire vascular tree were removed, sectioned, processed, and stained with hematoxylin and eosin. The entire vascular tree was also stained with Movat's pentachrome and oil red O (ORO) and assessed semiquantitatively (0 to 5+/5+) and quantitatively (mean intimal area and ORO positivity, mm2). Both blood lipid measurements (total cholesterol, high-density lipoprotein-cholesterol, triglycerides, and low-density lipoprotein-cholesterol) and vessel wall lesion assessment (intimal area, mm2) were statistically elevated (p < 0.05) in the cholesterol-fed cats as compared to those on a normal diet. The highest correlations obtained between blood lipid components and vessel wall measures were the percent increase in triglyceride from base line versus the ORO measurement or foam cell grade (r = 0.86), and percent increase in triglycerides versus the intimal area in the lower abdominal aorta (r = 0.91). Similar relationships were found when the intimal area in the brachiocephalic/subclavian vessels was correlated with the absolute triglyceride values (r = 0.85) or with the percent increase in triglycerides (r = 0.83). Thus, we produced atherosclerotic lesions in the cat within 2 to 4 months on a cholesterol-enriched diet; blood lipid levels were highly correlated with lesional measurements in the vessel wall. This study will provide the basis for evaluation of the susceptibility of New Zealand lipoprotein lipase-deficient cats to diet-induced atherosclerosis.

Plasma and vessel wall lipoprotein lipase have different roles in atherosclerosis.

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism, and has been hypothesized to exert either pro- or anti-atherogenic effects, depending on its localization. Decreased plasma LPL activity is associated with the high triglyceride (TG);-low HDL phenotype that is often observed in patients with premature vascular disease. In contrast, in the vessel wall, decreased LPL may be associated with less lipoprotein retention due to many potential mechanisms and, therefore, decreased foam cell formation. To directly assess this hypothesis, we have distinguished between the effects of variations in plasma and/or vessel wall LPL on atherosclerosis susceptibility in apoE-deficient mice. Reduced LPL in both plasma and vessel wall (LPL(+/-)E(-/-)) was associated with increased TG and increased total cholesterol (TC) compared with LPL(+/+)E(-/-) sibs. However despite their dyslipidemia, LPL(+/-)E(-/-) mice had significantly reduced lesion areas compared to the LPL(+/+)E(-/-) mice. Thus, decreased vessel wall LPL was associated with decreased lesion formation even in the presence of reduced plasma LPL activity. In contrast, transgenic mice with increased plasma LPL but with no increase in LPL expression in macrophages, and thus the vessel wall, had decreased TG and TC and significantly decreased lesion areas compared with LPL(+/+)E(-/-) mice. This demonstrates that increased plasma LPL activity alone, in the absence of an increase in vessel wall LPL, is associated with reduced susceptibility to atherosclerosis. Taken together, these results provide in vivo evidence that the contribution of LPL to atherogenesis is significantly influenced by the balance between vessel wall protein (pro-atherogenic) and plasma activity (anti-atherogenic).

ABCA1 regulatory variants influence coronary artery disease independent of effects on plasma lipid levels.

The authors have previously shown that individuals heterozygous for ABCA1 mutations have decreased high density lipoprotein cholesterol, increased triglycerides and an increased frequency of coronary artery disease (CAD), and that single nucleotide polymorphisms (SNPs) in the coding region of the ABCA1 gene significantly impact plasma lipid levels and the severity of CAD in the general population. They have now identified several SNPs in non-coding regions of ABCA1 which may be important for the appropriate regulation of ABCA1 expression (i.e. in the promoter, intron 1 and the 5' untranslated region), and have examined the phenotypic effects of these SNPs in the REGRESS population. Out of 12 SNPs, four were associated with a clinical outcome. A threefold increase in coronary events with an increased family history of CAD was evident for the G-191C variant. Similarly, the C69T SNP was associated with a twofold increase in events. In contrast, the C-17G was associated with a decrease in coronary events and the InsG319 was associated with less atherosclerosis. For all these SNPs, the changes in atherosclerosis and CAD occurred without detectable changes in plasma lipid levels. These data suggest that common variation in non-coding regions of ABCA1 may significantly alter the severity of atherosclerosis, without necessarily influencing plasma lipid levels.

Ethnic variation and in vivo effects of the -93t-->g promoter variant in the lipoprotein lipase gene.

Recently, a (t-->g) transition at nucleotide -93 in the lipoprotein lipase (LPL) gene promoter has been observed in Caucasians. Here, we have compared the frequency of the -93g carriers in three distinct populations (Caucasians, South African Blacks, and Chinese). The carrier frequency in the Caucasian population was 1.7% (4/232), which was in contrast to the South African Black population, which had a frequency for this allele of 76.4% (123/161) of the individuals tested. This transition was not observed in the Chinese population under study. Near complete linkage disequilibrium between the -93g and the previously described D9N mutation was observed in the Caucasian population but not in South African Blacks. To further assess the ancestral origins of these DNA changes, DNA haplotyping using a CA repeat 5' to these substitutions was performed. The -93t allele was associated with only a few specific dinucleotide repeat sizes. In contrast, the -93g allele occurred on chromosomes with many different repeat lengths. The broad distribution of repeats on -93g carrying chromosomes, their high frequency in the South African Black population, and the conservation of the -93g allele among different species may suggest that the -93g allele is the ancestral allele on which a transition to t and the D9N mutations arose. The very high frequency of the -93g allele distinct from the N9 allele in a cohort of Black South Africans allowed us to specifically assess the phenotypic effects of the -93g allele on lipids. Individuals homozygous for the g allele at -93 showed mildly decreased triglycerides compared with individuals homozygous for the t allele (1.14 +/- 0.66 mmol/L versus 0.82 +/- 0.3; P = .04). Thus, the -93g allele in this cohort is associated with low plasma triglyceride levels.

Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and ApoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1.

By using BAC transgenic mice, we have shown that increased human ABCA1 protein expression results in a significant increase in cholesterol efflux in different tissues and marked elevation in high density lipoprotein (HDL)-cholesterol levels associated with increases in apoAI and apoAII. Three novel ABCA1 transcripts containing three different transcription initiation sites that utilize sequences in intron 1 have been identified. In BAC transgenic mice there is an increased expression of ABCA1 protein, but the distribution of the ABCA1 product in different cells remains similar to wild type mice. An internal promoter in human intron 1 containing liver X response elements is functional in vivo and directly contributes to regulation of the human ABCA1 gene in multiple tissues and to raised HDL cholesterol, apoAI, and apoAII levels. A highly significant relationship between raised protein levels, increased efflux, and level of HDL elevation is evident. These data provide proof of the principle that increased human ABCA1 efflux activity is associated with an increase in HDL levels in vivo.

Mutations in the ABC1 gene in familial HDL deficiency with defective cholesterol efflux.

BACKGROUND: A low concentration of HDL cholesterol is the most common lipoprotein abnormality in patients with premature atherosclerosis. We have shown that Tangier disease, a rare and severe form of HDL deficiency characterised by a biochemical defect in cellular cholesterol efflux, is caused by mutations in the ATP-binding-cassette (ABC1) gene. This gene codes for the cholesterol-efflux regulatory protein (CERP). We investigated the presence of mutations in this gene in patients with familial HDL deficiency. METHODS: Three French-Canadian families and one Dutch family with familial HDL deficiency were studied. Fibroblasts from the proband of each family were defective in cellular cholesterol efflux. Genomic DNA of each proband was used for mutation detection with primers flanking each exon of the ABC1 gene, and for sequencing of the entire coding region of the gene. PCR and restriction-fragment length polymorphism assays specific to each mutation were used to investigate segregation of the mutation in each family, and to test for absence of the mutation in DNA from normal controls. FINDINGS: A different mutation was detected in ABC1 in each family studied. Each mutation either created a stop codon predicted to result in truncation of CERP, or altered a conserved aminoacid residue. Each mutation segregated with low concentrations of HDL-cholesterol in the family, and was not observed in more than 500 control chromosomes tested. INTERPRETATION: These data show that mutations in ABC1 are the major cause of familial HDL deficiency associated with defective cholesterol efflux, and that CERP has an essential role in the formation of HDL. Our findings highlight the potential of modulation of ABC1 as a new route for increasing HDL concentrations.

Lipid and lipoprotein analysis of cats with lipoprotein lipase deficiency.

BACKGROUND: We have previously described a colony of domestic cats with a naturally occurring mutation in the lipoprotein lipase (LPL) gene. We have now further characterized cats homozygous for LPL deficiency (LPL -/-, homozygotes), and have contrasted these with heterozygotes (LPL +/-) and normal cats (LPL +/+). MATERIALS AND METHODS: Density gradient ultracentrifugation with subsequent lipid analysis, agarose and polyacrylamide gel electrophoresis was used to examine detailed liproprotein differences between the genotypes. Oral fat loading studies and breast milk fatty acid analysis were also performed to further characterize the phenotypic expression of LPL deficiency in this model system. RESULTS: Several lipid abnormalities associated with homozygosity for LPL deficiency were evident. Triglyceride-rich lipoprotein-triglycerides (TRL-TG) and cholesterol (TRL-C) were higher (TRL-TG 2.09 +/- 1.14 vs. 0.15 +/- 0.04 mmol L-1, P < 0.001; TRL-C 0.42 +/- 0.30 vs. 0.11 +/- 0.16 mmol L-1, P < 0.05) in male -/- than in male +/+ cats, as was HDL-cholesterol (HDL-C, 1.75 +/- 0.24 vs. 1.41 +/- 0.14 mmol L-1, P < 0.05). LDL-C levels were lower in homozygous cats than in control cats, similar to what is seen in human LPL deficiency. Oral fat loading studies revealed that homozygous cats have a marked reduced ability to clear plasma TGs in terms of peak time (7 h vs. 3 h), peak height (9.36 vs. 1.1 mmol L-1), area under the TG clearance curve (AUC, 280.3 vs. 2.2 h mmol L-1) and time to return to baseline. Fasting lipid and lipoprotein levels were not significantly different between heterozygous and normal cats. However, oral fat loading in heterozygotes revealed an intermediate phenotype (peak of 2.35 mmol L-1 at 5 h, AUC 13.1 h mmol L-1), highlighting the impaired TG clearance in these animals. CONCLUSION: Thus, LPL deficiency in the cat results in a lipid and lipoprotein phenotype that predominantly parallels human LPL deficiency, further validating the use of these animals in studies on the pathobiology of LPL.