Original Article

Vitamin A is linked to the expression of the AI-CIII-AIV gene cluster in familial combined hyperlipidemia

Correspondence to: Josep Ribalta

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

There is growing evidence of the capacity of vitamin A to regulate the expression of the genetic region that encodes apolipoproteins (apo) A-I, C-III, and A-IV. This region in turn has been proposed to modulate the expression of hyperlipidemia in the commonest genetic form of dyslipidemia, familial combined hyperlipidemia (FCHL). The hypothesis tested here was whether vitamin A (retinol), by controlling the expression of the AI-CIII-AIV gene cluster, plays a role in modulating the hyperlipidemic phenotype in FCHL. We approached the subject by studying three genetic variants of this region: a C₁₁₀₀–T transition in exon 3 of the apoC-III gene, a G₃₂₀₆–T transversion in exon 4 of the apoC-III gene, and a G_-75–A substitution in the promoter region of the apoA-I gene. The association between plasma vitamin A concentrations and differences in the plasma concentrations of apolipoproteins A-I and C-III based on the different genotypes was assessed in 48 FCHL patients and 74 of their normolipidemic relatives. The results indicated that the subjects carrying genetic variants associated with increased concentrations of apoA-I and C-III (C₁₁₀₀–T and G_-75–A) also presented increased plasma concentrations of vitamin A. This was only observed among the FCHL patients, which suggested that certain characteristics of these patients contributed to this association. The G₃₂₀₆–T was not associated with changes in either apolipoprotein concentrations or in vitamin A.

In summary, we report a relationship between genetically determined elevations of proteins of the AI-CIII-AIV gene cluster and vitamin A in FCHL patients. More studies will be needed to confirm that vitamin A plays a role in FCHL which might also be important for its potential application to therapeutical approaches.—Ribalta, J., J. Girona, J. C. Vallvé, A. E. La Ville, M. Heras, and L. Masana. Vitamin A is linked to the expression of the AI-CIII-AIV gene cluster in familial combined hyperlipidemia. J. Lipid Res. 1999. 40: 426– 431.

Supplementary key words: peroxisome proliferator activated receptor, retinoid X receptor, retinoic acid, hyperapoB, fibrates, syndrome X

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Familial combined hyperlipidemia (FCHL) is a common genetic lipid disorder which is strongly associated with an increased risk of cardiovascular disease (1) (2). It is characterized by high concentrations of plasma cholesterol and/or triglycerides (3) which are postulated to result from hepatic overproduction of apolipoprotein (apo) B-100 particles (4) (5). However, the exact primary genetic defect in FCHL is not known. Lipoprotein profiles may vary in the patient over time, and different hyperlipidemic phenotypes are also present among the first degree relatives (6), implying that family studies are necessary if a correct diagnosis is to be made. There is considerable phenotypic overlap between FCHL and other lipid and metabolic disorders such as defective lipoprotein lipase (LPL) function (7), glucose intolerance, insulin resistance (8), or hypertension (9). Because this phenotypic heterogeneity has both environmental and genetic bases, a number of studies have attempted to identify these accompanying modifying factors.

The AI-CIII-AIV gene cluster appears to be an important region in modulating the expression of hyperlipidemia in FCHL (10) (11) (12). Dallinga-Thie et al. (10) reported a specific modifying effect of the AI-CIII-AIV gene cluster on plasma triglyceride and low density lipoprotein (LDL) cholesterol concentrations in these patients. This observation was confirmed when the same group showed that specific haplotypes of this genetic region made patients more susceptible to developing hyperlipidemia (12). Our group added that this region not only affected the lipid content of the lipoproteins but also the number of these particles which is relevant in view of the existing apoB-100 hepatic overproduction (11).

The mechanism by which the AI-CIII-AIV gene cluster influences the expression of the FCHL phenotype is currently not known. However, biochemical characteristics of FCHL, such as high plasma apoC-III concentrations (13), suggest that the control of the expression of these genes plays a major role.

Several studies indicate that vitamin A is an important regulator of the expression of the AI-CIII-AIV gene cluster (14) (15) (16) (17). Animal studies have shown that vitamin A modulates lipid transport in normal rats by altering apolipoprotein levels (14) and this effect has been confirmed in vitamin A-deficient rats (15). On the other hand, studies exploring the mode of action of certain drugs such as fibrates have shown that the expression of the apoA-I and C-III genes is controlled by a class of proteins that belong to the nuclear receptor superfamily, called peroxisome proliferator activated receptors (PPARs), and that heterodimerize with the retinoid X receptor (RXR). PPARs are activated by multiple stimuli directly resulting from the action of hypolipidemic agents (such as fibrates), diet, or lipid and glucose metabolism while RXR is activated by retinoic acid, an intracellular active form of dietary vitamin A (16) (17). The reported differences in the plasma concentrations of vitamin A between FCHL patients and normolipidemic controls also suggest that vitamin A has a role in FCHL (18).

Putting all these data together we hypothesized that vitamin A, by controlling the expression of the AI-CIII-AIV gene cluster, might play a role in FCHL. The aim of the present study was to explore whether variations in the AI-CIII-AIV gene cluster, which are associated with an increased expression of apolipoproteins A-I and C-III in patients with familial combined hyperlipidemia, are also associated with differences in plasma vitamin A concentrations in these patients. Three genetic variants of this region were selected: the C₁₁₀₀–T polymorphism in exon three of the apoC-III gene was selected because it has a frequency which is nearly double in FCHL patients than in controls (11) (19) and it is associated with higher concentrations of apoC-III and also apoA-I in such patients (11). The G_-75–A substitution in the promoter of the apoA-I gene was chosen because it is in very strong linkage disequilibrium with the XmnI RFLP (19) which has shown linkage with the FCHL phenotype in a number of families (20) and has been reported to be consistently associated with elevated concentrations of apoA-I (21). Finally, the G₃₂₀₆–T transversion in the non-translated region of exon 4 of the apoC-III gene was selected because it has been reported to present with increased frequency in combined hyperlipidemia (19) but is not associated with increased concentrations of either apoC-III or apoA-I.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Subjects
All subjects recruited for the study gave fully informed written consent and the protocol was approved by the Scientific and Ethical Committee of the Hospital Universitari de Sant Joan.

Sixteen FCHL families were identified by the following criteria: an index patient with plasma concentrations of cholesterol (chol) 6.4 mmol/L and triglycerides (trig) 2.8 mmol/L detected at any time in the clinical history and at least one first degree relative with a hyperlipoproteinemic phenotype different from that of the index patient. All available family members of index patients were recruited, a total of 106 individuals. Of the 122 subjects, those with plasma cholesterol and/or triglyceride concentrations 6.4 mmol/L and 2.8 mmol/L, respectively, or above the 95^th percentile for age and gender were included in the FCHL group (n = 48). Relatives who did not meet this criteria (n = 74) were assigned to the normolipidemic relatives (NL) group.

Biochemical analyses were conducted to rule out secondary causes of hyperlipidemia and apoE genotyping was performed to exclude type III hyperlipoproteinemia.

Analytical methods
A 10-ml venous blood sample was withdrawn after an overnight fast of 12 h. Triglyceride and cholesterol concentrations were measured using enzymatic kits (Boehringer Mannheim, Germany) adapted for a Cobas Mira centrifugal analyzer (Roche Pharmaceuticals, Switzerland) with Precinorm^® (Boehringer Mannheim, Germany) as quality control. Immunoturbidometry was used for the apolipoproteins using specific antiserum for apoA-I and apoB-100 (Boehringer Mannheim, Germany) and apoC-III (Daichii Chemicals, Japan). Low density lipoproteins were separated by sequential preparative ultracentrifugation (22) using a Kontron 45.6 fixed-angle rotor in a Centrikon 75 (Kontron Instruments, Italy). Total high density lipoprotein (HDL) cholesterol was measured after precipitation with polyethylene glycol (Immuno AG, Austria).

Vitamin A analyses
Vitamin A in plasma was measured using the method of Bieri, Tolliver, and Catignani (23). Briefly, vitamin A was extracted from 100 µl of plasma into n-hexane and 100 µl of a solution of all-trans-retinyl acetate (Sigma Chemical Co., St. Louis, MO) in ethanol was added as internal standard. The amount of retinyl acetate lost during the extraction was used to estimate how much retinol was lost during the procedure. The samples were centrifuged and 150 µL of the hexane layer was removed and evaporated under a stream of nitrogen. The reconstituted lipid residue was analyzed by high pressure liquid chromatography (HPLC) using the Hewlett-Packard 1050 series system (Hewlett-Packard GmbH, Germany) in which the separation column was Spherisorb ODS 2 and the mobile phase was methanol–water 98:2 (v/v). A standard curve of all-trans -retinol (Sigma Chemical Co.) was used to determine vitamin A concentrations. Absorbancies were recorded on a UV variable wavelength detector set at 325 nm.

DNA analyses
DNA was extracted from an aliquot of frozen white cells by the salting out method (24). The genetic variants in the AI-CIII-AIV gene cluster studied were: a C₁₁₀₀–T transition in exon 3 of the apoC-III gene, a G₃₂₀₆–T transversion in exon 4 of the apoC-III gene, and a G_-75–A substitution in the promoter region of the apoA-I gene. The region containing each of the variants was amplified separately by PCR in a reaction mixture that contained 250 ng of each of the two primers, 200 ng of genomic DNA, 1.5 mM MgCl₂, and 1 U of Taq polymerase (Boehringer Mannheim, Germany). The reactions were performed on a Hybaid Omnigene thermocycler at 95°C for 5 min, 55°C for 1 min, 72°C for 2 min followed by 35 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min. Specific primers were: 5' primer, 5'-CA ATGGGTGGTCAAGCAGAAGC-3' and 3' primer, 5'-GAGCACCT CCATTCCATTGTTGG-3' (for C₁₁₀₀–T); 5' primer, 5'-CATGGT TGCCTACAGAGGATG-3' and 3' primer, 5'-TGACCTTCCGC ACAAAGCTGT-3' (for G₃₂₀₆–T); and 5' primer, 5'-AGGGACAGA GCTGATCCTTGAACTCTTAAG-3' and 3' primer, 5'-TTAGGGG ACACCTAGCCC TCAGGAAGAGCA-3' (for G_-75–A). For the C₁₁₀₀–T and G₃₂₀₆–T genotypes, PCR products were run on a 2% agarose gel and double-blotted on Hybond-N⁺ membrane (Amersham International, U.K.) and subsequently hybridized with the following oligonucleotides for C₁₁₀₀–T: C allele 5'-ATG CAGGGCTACATGAA-3' and T allele 5'-TTCATGTAACCCTG CAT-3'; for G₃₂₀₆–T: G allele: 5'-ACAGGGGCAGCCCTG-3' and T allele 5'- CAGGGCTTCCCCTGT-3' according to the conditions described elsewhere (25). For the G_-75–A polymorphism, 20 µL of PCR product was digested overnight with 9 U of the restriction enzyme MspI (Boehringer Mannheim, Germany) in a total volume of 30 µL using the buffer recommended by the manufacturer. The DNA fragments were separated by electrophoresis on 2% agarose gels. Digestion with MspI resulted in four fragments of 48, 67, 110, and 207 bp. In the absence of the MspI cutting site, three fragments of 48, 177, and 207 bp were produced (26).

Statistical analyses
The chi-square (²) test was used to compare the frequency of the genotypes among groups and to test for the Hardy-Weinberg equilibrium. The Z-test for comparison of proportions was used to compare allele frequencies that were determined by gene counting. ANOVA was performed to compare the means of lipid, lipoprotein, apolipoprotein, and vitamin A data adjusted for age and gender and BMI and log transformed when the variables were not normally distributed. Comparisons using unadjusted data were made with the Student's t -test. Results are expressed as mean (standard deviation). Statistical significance was accepted at the 0.05 level. Analyses were carried out with the SPSS 7.5. 2S version statistical package.

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

The FCHL and NL groups had a similar male/female distribution. The FCHL group presented significantly higher age, BMI, and blood pressure values ( Table 1). Plasma lipids, lipoproteins and apolipoproteins, including apoA-I, were significantly elevated in the FCHL group with the exception of HDL chol which was not different between groups ( Table 2). Plasma cholesterol/triglyceride ranges in the FCHL and NL groups were (4.28–8.35 mmol/L)/(0.50–3.49 mmol/L) and (2.45–6.12 mmol/L)/(0.36 –2.52 mmol/L), respectively.

Plasma vitamin A concentrations
Plasma vitamin A concentrations were not different between the FCHL (527.5 (240.3) µg/L) and the normolipidemic (489.6 (335.9) µg/L) groups ( Figure 1). Table 3 shows the correlation between plasma vitamin A levels and lipids, lipoproteins and apolipoproteins which were very low in the two groups although in some cases they reached statistical significance. The correlation between vitamin A and apolipoproteins A-I and C-III gave a correlation coefficient (r) of 0.23 and 0.30 for the FCHL group and 0.07 and 0.26 for the NL group, respectively. Correlations were just as low, when all subjects were analyzed together, between vitamin A and plasma cholesterol (r = 0.18), plasma triglycerides (r = 0.19), LDL chol (r = 0.16) and apoB-100 (r = 0.24). When the two groups were studied separately, statistical significance was only maintained in the normolipidemic group, most probably due to the size of the sample. Linear regression analysis indicated that neither cholesterol nor triglyceride accounted for a significant percentage of the vitamin A variability in this sample. All this indicated a lack of association between the variations in vitamin A and lipid, lipoprotein and apolipoprotein concentrations.

View larger version (36K): [in this window] [in a new window]   Figure 1. Box-plot representation of plasma vitamin A concentrations (µg/L) in FCHL patients (FCHL) and their normolipidemic relatives (NL).

G_-75–A polymorphism in the apoA-I gene
The frequencies of the GG, GA, and AA genotypes were not different from those predicted by the Hardy-Weinberg distribution. The A_-75 allele and the GA/AA genotype frequencies were not different between the FCHL (0.18 and 0.31, respectively) and the normolipidemic group (0.17 and 0.32, respectively). Due to the low frequency of the minor allele, all carriers of the A_-75 (heterozygotes and homozygotes) were pooled for statistical analysis. The association between the G_-75–A polymorphism and vitamin A, apoA-I, and apoC-III, investigated in all the subjects, showed that the GA/AA genotype was associated with increased concentrations of apoA-I (P = 0.010), apoC-III (P = 0.031), and with elevations of vitamin A which almost reached statistical significance (P = 0.054) ( Table 4). When the two groups were studied separately it was observed that the GA/AA genotype was associated with elevated concentrations of apoA-I (P = 0.002), apoC-III (P = 0.017), and vitamin A (P = 0.015) in the FCHL group while none of these associations was detected among the normolipidemic subjects.

The A_-75 allele was associated with significantly higher plasma cholesterol (P = 0.040) and elevated plasma triglyceride (P = 0.054) in the FCHL group.

C₁₁₀₀–T and G₃₂₀₆–T polymorphisms in the apoC-III gene
The frequencies of the genotypes for both polymorphisms were not different from those predicted by the Hardy-Weinberg distribution. The T₁₁₀₀ allele and CT/TT genotype frequencies of the C₁₁₀₀–T polymorphism were not different between the FCHL (0.33 and 0.54, respectively) and the normolipidemic (0.26 and 0.45, respectively) groups. Carriers of the T₁₁₀₀ alllele were pooled for statistical analyses. When all subjects were considered together, the CT/TT carriers had significantly higher concentrations of apoA-I (P = 0.024) and apoC-III (P = 0.047). This was only observed among the affected subjects when the groups were analyzed separately. More importantly, only the FCHL subjects also presented significantly higher concentrations of vitamin A (P = 0.043) ( Table 5). The T₁₁₀₀ allele was associated with significantly higher plasma triglyceride (P = 0.028) in the FCHL group.

The allelic frequency of the minor T₃₂₀₆ allele in the FCHL group (0.42) was no different from that of the normolipidemic group (0.37). As this was a fairly common variation, genotypes were not grouped for association analysis and, therefore, GG, TG, and TT genotypes were considered separately. This polymorphism showed no association with either apoA-I or apoC-III plasma concentrations in any of the groups studied. However, a significant association with higher plasma vitamin A concentrations in the subjects carrying the TT genotype (P = 0.009) was detected ( Table 6). Separate analysis of the FCHL and NL groups indicated that this association was confined to the group of normolipidemic relatives. There were no significant differences between the genotypes with regard to plasma cholesterol or triglyceride.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Our hypothesis was that vitamin A influences the hyperlipidemic phenotype in FCHL by contributing to the control of the expression of the AI-CIII-AIV gene cluster. We approached the subject by studying genetically determined elevations of apolipoproteins A-I and C-III and their relation to plasma vitamin A concentrations in FCHL patients.

Our results indicate that, among FCHL subjects, the genetic variants in the AI-CIII-AIV gene cluster associated with higher plasma concentrations of apoA-I and C-III are also associated with higher vitamin A levels. This is physiologically important because it can be observed in the group of patients but not in their unaffected relatives which suggests that characteristics of these patients contribute to the relationship between the AI-CIII-AIV gene cluster and vitamin A.

These results, although indicative of the possibility that vitamin A plays a role in FCHL expression, do not provide evidence that vitamin A directly controls the expression of this genetic region. Other factors such as lipemia are important determinants of the variability of these two proteins. In the present study the A_-75 and the T₁₁₀₀ alleles were associated with increased cholesterol and triglyceride in plasma, respectively. Moreover, linear regression analysis indicated that 65% of the variability of plasma apoC-III was explained by total triglyceride whereas 59% of the variation of apoA-I was explained by cholesterol in the HDL fraction (data not shown). However, plasma lipids or lipoproteins did not explain a significant percentage of the variability observed in plasma vitamin A. This is not surprising as the vitamin A measured in this study is not associated with lipoproteins (as would be the case for carotenoids or retinyl esters), but is mainly bound to retinol binding protein and is very tightly regulated by the liver. Therefore the nature of these variations associated with genetic variants is somewhat intriguing and suggests that vitamin A has an indirect relationship with the AI-CIII-AIV gene cluster which, based on the results of linear regression analysis, is probably non-mediated by lipoproteins. This lack of a direct asociation between vitamin A and apolipoproteins A-I and C-III and vitamin A strongly suggests that other factors also contribute to the observed effect.

One crucial aspect is to elucidate by which mechanism vitamin A is capable of regulating the expression of certain proteins at a genetic level. Pharmacological studies that have investigated the mode of action of fibrates have partly responded to this (16) (17). Fibrates were found to activate the transcription factor PPAR which forms a heterodimer with another transcription factor RXR. The active heterodimer recognizes response elements of certain genes (such as apoA-I and C-III) and controls their expression. In the context of vitamin A it is important to note that RXR must be activated by retinoic acid, the intracellular active form of vitamin A. Most of the intracellular retinoic acid derives from plasma vitamin A which is 100 times more abundant, which may mean that the availability of vitamin A is important for the efficiency of this system. This point has actually been confirmed by Mukherjee et al. (27) who showed that substances with vitamin A activity that are also agonists of RXR can activate PPAR-inducible genes and promote a more efficient effect of fibrates. In the light of this evidence, the intracellular concentrations of retinoic acid, PPAR or RXR appear as potential candidates for the observed association between plasma vitamin A concentrations and apolipoproteins A-I and C-III.

In summary we report a relationship between increased plasma concentrations of apolipoproteins A-I and C-III and vitamin A in FCHL patients that is not mediated by the accompanying hyperlipidemia. More studies will be needed to confirm that vitamin A has a role in FCHL, which might be relevant also for its potential application to therapeutical approaches.

	ACKNOWLEDGMENTS

The authors wish to thank Prof. Steve Humphries and Dr. Chung-Fang Xu for invaluable help and advice in the setting up of the molecular biology techniques; Silvia Olivé, for her excellent technical support; Drs. Núria Plana, Pilar Sardà, Rosa Solà, and Carlos Alonso-Villaverde for their clinical assistance. This study has been supported, in part, by the Fundació Joan Abelló Pascual and the Ministerio de la Salud (DGICYT # PM 92- 0209).

Manuscript received July 13, 1998; and in revised form October 29, 1998.

Abbreviations: FCHL, familial combined hyperlipidemia; VLDL, very low density lipoprotein; LDL, low density lipoprotein; PPAR, peroxisome proliferator activated receptor; RXR, retinoid X receptor; HPLC, high pressure liquid chromatography; HDL, high density lipoprotein; RFLP, restriction fragment length polymorphism

	REFERENCES

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

1. Rose, H. G., Kranz, P., Weinstock, M., Juliano, J., Haft, J. I. 1973. Inheritance of combined hyperlipoproteinemia: evidence for a new lipoprotein phenotype. Am. J. Med. 54:148-160[Medline].

2. Goldstein, J. L., Schrott, H. G., Hazzard, W. R., Bierman, E. L., Motulsky, A. G. 1973. Hyperlipidaemia in coronary heart disease. II. Genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidemia. J. Clin. Invest. 52:1544-1568.

3. Nikkilä, E. A., Aro, A. 1973. Family study of serum lipids and lipoproteins in coronary heart disease. Lancet. 1:954-959[Medline].

4. Kwiterovich, P. O., Jr., White, S., Forte, T., Bachoricik, P. S., Smith, H., Sniderman, A. 1987. Hyperapobetalipoproteinemia in a kindred with familial combined hyperlipidemia and familial hypercholesterolemia. Arteriosclerosis. 7:211-225[Abstract/Free Full Text].

5. Sniderman, A., Brown, B. G., Stewart, B. F., Cianflone, K. 1992. From familial combined hyperlipidemia to hyperapoB: unravelling the overproduction of hepatic apolipoprotein B. Curr. Opin. Lipidol. 3:137-142.

6. Brunzell, J. D., Albers, J. J., Chait, A., Grundy, S. M., Groszek, E., McDonald, G. B. 1983. Plasma lipoproteins in familial combined hyperlipidemia and monogenic familial hypertriglyceridemia. J. Lipid Res. 24:147-155[Abstract].

7. Babirak, S. P., Brown, G., Brunzell, J. D. 1992. Familial combined hyperlipidemia and abnormal lipoprotein lipase. Arterioscler. Thromb. 12:1176-1183[Abstract].

8. Reaven, G. M. 1988. Banting lecture 1988. Role of insulin resistance in human disease. Diabetes. 37:1595-1607[Abstract].

9. Hunt, S. C., Wu, L. L., Hopkins, P. N., Stults, B. M., Kuida, H., Ramirez, M. E., Lalouel, J. M., Williams, R. R. 1989. Apolipoprotein, low density lipoprotein subfraction, and insulin associations with familial combined hyperlipidemia (Study of Utah patients with familial dyslipidemic hypertension). Arteriosclerosis. 9:335-344[Abstract/Free Full Text].

10. Dallinga-Thie, G. M., Bu, X. D., v L-S. Trip, M., Rotter, J. I., Lusis, A. J., de Bruin, T. W. A. 1996. Apolipoprotein A-I/C-III/A-IV gene cluster in familial combined hyperlipidemia: effects on LDL-cholesterol and apolipoproteins B and C-III. J. Lipid Res. 37:136-147[Abstract].

11. Ribalta, J., La Ville, A. E., Vallvé, J. C., Humphries, S., Turner, P. R., Masana, L. 1997. A variation in the apolipoprotein C-III gene is associated with an increased number of circulating VLDL and IDL particles in familial combined hyperlipidemia. J. Lipid Res. 38:1061-1069[Abstract].

12. Dallinga-Thie, G. M., v L-S. Trip, M., Rotter, J. I., Cantor, R. M., Bu, X. D., Lusis, A. J., de Bruin, T. W. A. 1997. Complex genetic contribution of the AI-CIII-AIV gene cluster to familial combined hyperlipidemia. J. Clin. Invest. 99:953-961[Medline].

13. Castro Cabezas, M., de Bruin, T. W. A., Jansen, H., Kock, A. W., Kortlandt, W., Erkelens, D. W. 1993. Impaired chylomicron remnant clearance in Familial Combined Hyperlipidemia. Arterioscler. Thromb. 13:804-814[Abstract/Free Full Text].

14. Nagasaki, A., Kikuchi, T., Kurata, K., Masushige, S., Hasegawa, T., Kato, S. 1994. Vitamin A regulates the expression of the apolipoprotein AI and CIII genes in the rat. Biochem. Biophys. Res. Commun. 205:1510-1571[Medline].

15. Zolfaghari, R., Ross, A. C. 1994. Effect of vitamin A deficiency and retinoic acid repletion on intestinal and hepatic apolipoprotein A-I mRNA levels of adult rats. J. Lipid Res. 35:1985-1992[Abstract].

16. Auwerx, J., Schoonjans, K., Fruchart, J. C., Staels, B. 1996. Transcriptional control of triglyceride metabolism: fibrates and fatty acids change the expression of the LPL and apo C-III genes by activating the nuclear receptor PPAR. Atherosclerosis. 124:S29-S37.

17. Schoonjans, K., Staels, B., Auwerx, J. 1996. The peroxisome proliferator activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation. Biochim. Biophys. Acta. 1302:93-109[Medline].

18. Ribalta, J., La Ville, A. E., Girona, J., Vallvé, J. C., Masana, L. 1997. Low plasma vitamin A concentrations in familial combined hyperlipidemia. Clin. Chem. 43:2379-2383[Abstract/Free Full Text].

19. Xu, C. F., Talmud, P., Schuster, H., Houlston, R., Miller, G., Humphries, S. 1994. Association between genetic variation at the APO AI-CIII-AIV gene cluster and familial combined hyperlipidemia. Clin. Genet. 46:385-397[Medline].

20. Wojciechowski, A. P., Farrall, M., Cullen, P., Wilson, T. M. E., Bayliss, J. D., Farren, B., Griffin, B. A., Caslake, M. J., Packard, C. J., Shepherd, J., Thakker, R., Scott, J. 1991. Familial combined hyperlipidemia linked to the apolipoprotein AI-CIII-AIV gene cluster on chromosome 11q23–q24. Nature. 249:161-164.

21. Sigurdsson, G., Jr., Gudnason, V., Sigurdsson, G., Humphries, S. E. 1992. Interaction between a polymorphism of the apo A-I promoter region and smoking determines plasma levels of HDL and apo A-I. Arterioscler. Thromb. 12:1017-1022[Abstract/Free Full Text].

22. Schumaker, V. R., Puppione, D. L. 1986. Sequential flotation ultracentrifugation. Methods Enzymol. 128:155-170[Medline].

23. Bieri, J. C., Tolliver, T. J., Catignani, G. L. 1979. Simultaneous determination of alpha-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography. Am. J. Clin. Nutr. 10:2143-2149.

24. Miller, S. A., Dykes, D. D., Polesky, H. F. 1988. A simple salting out method procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16:1215[Free Full Text].

25. Xu, C. F., Talmud, P., Humphries, S. E. 1994. Three new polymorphisms of the apoAI-CIII-AIV gene cluster. Mol. Cell. Probes. 8:331-332[Medline].

26. Xu, C. F., Angelico, F., Del Ben, M., Humphries, S. 1993. Role of genetic variation at the apo AI-CIII-AIV gene cluster in determining plasma apo AI levels in boys and girls. Genet. Epidemiol. 10:113-122[Medline].

27. Mukherjee, R., Strasse, J., Jow, L., Hoener, P., Paterniti, J. R., Jr., Heyman, R. A. 1998. RXR agonists activate PPAR-inducible genes, lower triglycerides, and raise HDL levels in vivo. Arterioscler. Thromb. Vasc. Biol. 18:272-276[Abstract/Free Full Text].