Contribution of polymorphisms in the apolipoprotein AI-CIII-AIV cluster to hyperlipidaemia in patients with gout
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《研究生医学杂志》
1 Servicio de Endocrinología y Nutrición, Complejo Hospitalario Universitario Carlos Haya, Málaga, Spain
2 Servicio de Reumatologia, Hospital Reina Sofia, Cordoba, Spain
Background: Studies have shown that hyperuricaemia is independently related to the insulin resistance syndrome and that polymorphisms of the apolipoprotein AI-CIII-AIV cluster are also related to insulin resistance.
Objective: To study the prevalence of polymorphisms of the apolipoprotein AI-CIII-AIV cluster in persons with gout and to determine whether these polymorphisms contribute to the pathophysiology of gout or to altered lipid concentrations.
Methods: Plasma cholesterol, triglycerides, uric acid, VLDL, LDL, IDL, and HDL triglycerides, cholesterol, and the renal excretion of uric acid were measured in 68 patients with gout with gout and 165 healthy subjects. Polymorphisms were studied by amplification and RFLP in all subjects, using XmnI and MspI in the apolipoprotein AI gene and SstI in the apolipoprotein CIII gene.
Results: The A allele at position –75 bp in the apolipoprotein AI gene was more common in patients with gout than in controls (p = 0.01). Levels of cholesterol, triglycerides, uric acid, basal glycaemia, and HDL cholesterol were higher in the patients (p<0.001). In the patients there was also an interaction between mutations at the two polymorphic loci studied in the apolipoprotein AI gene (p = 0.04). An absence of the mutation at position –75 bp of the apolipoprotein AI gene resulted in increased plasma triglyceride levels.
Conclusions: Gouty patients have an altered allelic distribution in the apolipoprotein AI-CIII-AIV cluster, which could lead to changes in levels of lipoproteins. This is not caused by a single mutation but rather by a combination of different mutations.
Keywords: apolipoprotein AI-CIII-AIV cluster; gout; polymorphisms
Hyperuricaemia appears to be an independent risk factor for coronary heart disease.1–3 Although the reason for this is not well known, evidence suggests that hyperuricaemia forms part of the metabolic syndrome. A high concentration of uric acid is closely related to hyperinsulinaemia, glucose intolerance, hypertension, dyslipidaemia, very high levels of triglycerides, low levels of high density lipoprotein (HDL) cholesterol, and obesity (especially abdominal adiposity).4–12 Regarding the association that has already been demonstrated between hyperuricaemia and hyperlipidaemia, we have previously shown the existence of two groups of hyperuricaemic patients—those in whom the hyperuricaemia is associated with hyperlipidaemia, who could be considered to have the metabolic syndrome, and those having no association with any metabolic disorder.13 We have also reported that hyperuricaemic-hypertriglyceridaemic patients have high levels of very low density lipoprotein (VLDL) components, a reduced fractionated excretion of uric acid, and an increased ratio of CIII to CII apolipoproteins. Apolipoproteins therefore play an important role in the pathophysiology of the changes seen in hyperuricaemia.14
Apolipoproteins AI-CIII-AIV are lipid binding proteins that are involved in the transport of lipids in plasma. Defects or variations in these apolipoproteins are also associated with altered plasma concentrations of lipids and lipoproteins. The relation between variations in the apolipoprotein AI-CIII-AIV gene cluster and plasma lipids has long been recognised. Most studies have concentrated on the association between plasma triglycerides and variations in the gene cluster.15 Upregulation of apolipoprotein CIII in transgenic mice induces hypertriglyceridaemia because this apoprotein is involved in VLDL clearance.16 The apoprotein CIII gene is closely linked to the apolipoprotein AI and AIV genes on chromosome 11, forming a cluster. Study of polymorphisms in this cluster has shown that the S2 allele of the polymorphism is related to hypertriglyceridaemia and an increased risk for coronary heart disease.17,18 López-Miranda et al found that carriers of the S2 allele had reduced concentrations of LDL cholesterol when given a diet rich in monounsaturated fats.19 Furthermore, the change of a G for an A at position –75 base pairs (bp) in the promoter region of the apolipoprotein AI gene, digested by the restriction enzyme MspI, induces increased transcription of this gene and increased serum levels of apolipoprotein AI.20 Individuals with this mutation also have higher levels of HDL cholesterol. Although not yet demonstrated, it would appear that this can be explained by linkage disequilibrium between the A allele and another functional mutation in the gene. There is another mutation in the 5' region near the apolipoprotein AI gene –2500 bp upstream from the restriction start site, which is the marker for combined family hyperlipidaemia or hypertriglyceridaemia.21–23 These data show that the cluster of apolipoprotein AI-CIII-AIV genes is involved in hypertriglyceridaemia, and may also be involved in predisposition to atherosclerosis.
We studied the prevalence of polymorphisms of the apolipoprotein AI-CIII-AIV cluster in persons with gout and determined whether these polymorphisms contribute to the pathophysiology of hyperuricaemia or to altered lipid levels in these individuals.
Subjects
The study was undertaken in 68 men with gout attending the rheumatology service at the Hospital Reina Sofía in Cordoba, Spain. None was receiving lipid lowering or urate lowering treatment. Height and weight were measured and the body mass index (BMI) calculated.
Patients with secondary hyperuricaemia (renal failure or use of diuretics) were excluded from the study.
A control group was composed of 165 healthy persons with no history of gout and with cholesterol and triglyceride concentrations lower than 5.2 and 2.3 mmol/l, respectively, selected from a local cross sectional study24 and adjusted for age.
Procedures
DNA analysis
DNA was isolated from venous blood by the "salting out" method of Miller et al,25 modified by Queipo-Ortu?o et al,26 and amplified by polymerase chain reaction in a thermocycler (mastercycler Eppendorff). Primers for the XmnI locus (at position –2500 bp upstream of the transcription start site of the apolipoproteins AI) were those described by Shoulders et al,27 while those of Jeenah et al were used for the –75 bp locus of the apolipoprotein AI gene (determined by MspI),28 and those of Dammerman et al for the SstI locus of the apolipoprotein CIII gene.29
Laboratory analysis
Venous blood samples, collected after a 12 hour fast, were ultracentrifuged at 2000xg for 15 minutes for measurement of lipids and lipoproteins. Plasma levels of glucose, cholesterol, triglycerides, creatinine, HDL cholesterol, low density lipoprotein (LDL) cholesterol, and uric acid were measured in an autoanalyser using calorimetric assays (Ecoline 25, Diagnostica Merck). Calculations were made of uric acid clearance and the 24 hour uric acid urinary excretory fraction.
Lipoproteins were separated from serum by ultracentrifugation as follows: first, the VLDL fraction was separated from the serum by flotation at 125 000xg for 18 hours at 7°C in a 45° rotor (Beckman TLA 100.3) After separation of the VLDL, the density of the infranatant was adjusted to 1.30 g/ml by direct addition of potassium bromide and saccharose, separating the rest of the lipoproteins by density gradient centrifugation at 208 000xg for 22 hours at 7°C in a swinging bucket rotor (Beckman SW 40Ti). To visualise the position of the lipoprotein bands a small amount (25 μl) of saturated Coomassie blue was added. The concentrations of cholesterol and triglycerides were measured in each fraction as above. The amount of HDL cholesterol was also measured by the alternative method of precipitation with phosphotungstic acid in patients and controls. The LDL cholesterol was calculated from the Friedewald equation.
Statistical analysis
Data are expressed as mean (SD). Comparison between groups was made using Student’s t test for independent variables or the Mann–Whitney U test, depending on the normality of the distribution. Differences in the genotype distribution of the apolipoprotein AI-CIII-AIV cluster were studied by 2 tests. Statistical analyses were carried out with SPSS 6.0 for Windows and probability (p) values <0.05 were considered significant.
The clinical and laboratory data for the patients and controls are summarised in table 1. Plasma concentrations of cholesterol, triglycerides, and uric acid were higher in patients with gout than in the controls: (mean (SD) 5.48 (1.04) v 4.49 (0.45); 2.79 (2.51) v 1.01 0.45); and 0.46 (0.10) v 0.31 (0.07) mmol/l, respectively; p<0.001. The patients also had higher plasma HDL cholesterol levels (1.22 (0.32) v 1.08 (0.24) mmol/l; p<0.001) and a higher BMI (30.2 (3.8) v 27.3 (3.7) kg/m2; p<0.001) than the controls (table 1).
Frequency distribution of the polymorphisms of the apolipoprotein AI-CIII-AIV cluster
The frequency of the A allele—that is, the presence of the mutation in both homozygotes and heterozygotes—was higher in patients than in controls (p = 0.01). The distribution of the different genotypes of this polymorphism is shown in table 2. The unusual alleles in homozygotes located –2500 bp upstream of the transcription start site of the apolipoprotein AI gene (determined by XmnI) and of exon 4 of the apolipoprotein CIII gene (determined by SstI) were also more frequent in patients than controls, though the difference was not significant.
Influence of the polymorphisms of the apolipoprotein AI-CIII-AIV cluster on biological variables
The effect of these polymorphisms on the study variables differed. The presence of the X2 allele in the controls was not related to any increase in the variables studied, whereas its presence in the patients was related to an increased plasma concentration of triglycerides (2.31 (1.68) v 3.82 (3.76) mmol/l; p = 0.03) (table 3). The other polymorphisms studied in the apolipoprotein AI-CIII-AIV cluster were not related significantly to any differences in the distribution of the study variables (data not shown).
The content of VLDL triglycerides was greater in those patients with the X2 allele than in non-carriers, adjusted for age and BMI (1.77 (1.96) v 1.05 (0.71) mmol/l; p = 0.05) (table 3). The levels of IDL and LDL triglycerides were also higher in carriers of the mutated allele, though the difference was not statistically significant. There were no significant differences in any of the other lipoprotein fractions studied (table 3), or in the distribution of the biological variables in either of the other two polymorphisms of the apolipoprotein AI and CIII cluster (data not shown).
The combined distribution of the polymorphisms of the apolipoprotein AI-CIII-AIV cluster differed between the patients and the controls; 63.5% of the patients had some mutation at the two polymorphic sites of the apolipoprotein AI gene studied, compared with 41.7% of the controls (p = 0.007) (table 4). The presence of at least one unusual allele was related to significant differences in the lipid content of the lipoprotein fractions (table 5). VLDL cholesterol was higher in patients with at least one mutation in any of the polymorphisms of the cluster than in patients with no mutation (p = 0.044). The triglyceride content was greater in carriers of some mutation in the cluster than in persons with no mutation (p = 0.014). The IDL cholesterol was higher in carriers of some mutation than in non-carriers (p = 0.021). HDL cholesterol was also higher in carriers, but the difference was not significant.
Hyperuricaemia is a risk factor for coronary heart disease.30 The relation between hyperuricaemia-hyperlipidaemia and renal excretion of urates is known—individuals with hyperuricaemia-hyperlipidaemia have reduced renal excretion of urates compared with those who have only isolated hyperuricaemia; and the latter have also been reported to have low renal excretion of urates.31,32 In patients with hyperuricaemia and increased VLDL there is a close relation between the VLDL levels and renal excretion of urates.32,33 This relation appears to be mediated through the high prevalence of the E2 isoform of apolipoprotein E.34
Recent studies have related polymorphisms of the cluster to variations in plasma lipid levels. Variations in these genes contribute to combined family hyperlipidaemia, modifying plasma concentrations of cholesterol and triglycerides, as well as those of apolipoprotein B and apolipoprotein CIII.35
Patients with gout have a greater prevalence of mutated genotypes at polymorphic sites of the apolipoprotein AI gene; in our study, 18.8% had AA at position –75 bp from the transcription start site v 6.7% in controls (p = 0.01). This high prevalence may be responsible for the high levels of HDL cholesterol seen in patients with gout, as has been described for other disease groups.36–39
We also found a greater frequency of the mutated allele at position –2500 bp in the apolipoprotein AI gene, although the difference was not significant. We showed that 63.5% of persons with gout have some mutation at the two polymorphic sites of the apolipoprotein AI gene, compared with 36.7% who do not have these alleles (p = 0.007). These patients with a mutated allele at position –2500 bp upstream of the apolipoprotein AI gene had higher plasma and VLDL triglyceride levels than those without the mutation (p = 0.05), corrected for BMI (patients with the mutation had a lower BMI than those without the mutation). This allele has been associated with hypertriglyceridaemia in patients with combined family hyperlipidaemia.40,41 In our group of patients there seemed to be a synergic effect of the two polymorphic sites in the influence on plasma triglycerides. Patients with gout who had some mutation at the three polymorphic sites studied had higher contents of VLDL, intermediate density lipoprotein (IDL), HDL cholesterol, and VLDL triglycerides. Thus their metabolism of lipoproteins seems to be altered, thereby contributing to the expression of hypertriglyceridaemia. The lipoprotein phenotype characteristic of the insulin resistance syndrome was more prevalent in the gouty subjects with a mutation in the cluster despite their BMI being no greater than in subjects without the mutation.
We conclude that the high prevalence of mutations in the apolipoprotein AI-CIII-AIV cluster in patients with gout partly explains their lipoprotein phenotype and that, jointly, these mutations exert more influence on the polymorphic sites of the apolipoprotein AI gene than the apolipoprotein CIII gene.
ACKNOWLEDGEMENTS
This study was supported by grants from "Red de metabolismo y nutricion Fis C03/08" and a predoctoral grant from the Fundación Hospital Carlos Haya. We want to thank Pablo Rodriguez and Isabel Recio for excellent laboratory technical assistance, and gratefully acknowledge Ian Johnstone for his expertise in preparing the manuscript.
Gertler MM, Garn SM, Levine SA. Serum uric acid in relation to age and physique in health and coronary heart disease. Ann Intern Med 1951;34:1421–31.
Iribarren C, Sharp DS, Curb JD, Yano K. High uric acid: a metabolic marker of coronary heart disease among alcohol abstainers? J Clin Epidemiol 1996;49:673–8.
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Galvan AQ, Natali A, Baldi S, Frascerra S, Sanna G, Ciociaro D, Ferrannini E. Effect of insulin on uric acid excretion in humans. Am J Physiol 1995;268:E1–5.
Reaven GM, Lithell H, Landsberg L. Hypertension and associated metabolic abnormalities the role of insulin resistance and the sympathoadrenal system. N Engl J Med 1996;334:374–81.
Modan M, Halkin H, Karasik A, Lusky A. Elevated serum uric acid a facet of hyperinsulinemia. Diabetologia 1987;30:713–18.
Selby JV, Friedman GD, Quesenberry CP. Precursors of essential hypertension: Pulmonary function, heart rate, uric acid, serum cholesterol, and other chemistries. Am J Epidemiol 1990;131:1017–27.
Zavaroni I, Mazza S, Fantuzzi M, Dall’Aglio E, Bonora E, Delsignore R, et al. Changes in insulin and lipid metabolism in males with asymptomatic hyperuricemia. J Intern Med 1993;234:25–30.
Wyngaarden JB, Kelley WN. Gout. In: Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS, eds. The metabolic basis of inherited disease. New York: McGraw-Hill, 1983:1043–14.
Folsom AR, Burke GL, Ballew C, Jacobs DR, Haskell WL, Donahue RP, et al. Relation of body fatness and its distribution to cardiovascular risk factors in young blacks and whites. The role of insulin. Am J Epidemiol 1989;130:911–24.
Tinahones FJ, Vazquez F, C-Soriguer FJ, Collantes E. Lipoproteins in patients with isolated hyperuricemia. Adv Exp Med Biol 1998;431:61–7.
Tinahones FJ, Collantes E, C-Soriguer FJ, Gonzalez-Ruiz A, Pineda M, Anon J, et al. Increased VLDL levels and diminished renal excretion of uric acid in hyperuricaemic-hypertriglyceridaemic patients. Br J Rheumatol 1995;34:920–4.
Groenendijk M, Cantor RM, De Bruin TWA, Dallinga-Thie GM. New genetic variants in the apoA-I and apoC-III genes and familial combined hyperlipidemia. J Lipid Res 2001;42:188–94.
Waterworth DM, Ribalta J, Nicaud V, Dallongeville J, Humphries SE, Talmud P. ApoCIII gene variants modulate postprandial response to both glucose and fat tolerance tests. Circulation 1999;99:1872–7.
Ferns GAA, Ritchie C, Stocks J, Galton DJ. Genetic polymorphisms of apolipoprotein C-III and insulin in survivors of myocardial infarction. Lancet 1985;ii:300–3.
Ordovás JM, Civeira F, Genest JJ, Craig S, Robbins AH, Meade T, et al. Restriction fragment length polymorphisms of the apolipoprotein A-I, C-III and A-IV gene locus: relationships with lipids, apolipoproteins and premature coronary artery disease. Atherosclerosis 1991;87:75–86.
López Miranda J, Jansen S, Ordovás JM, Salas J, Marín C, Castro P, et al. Influence of the SstI polymorphic site at the apolipoprotein C-III gene on plasma LDL cholesterol response to monounsaturated dietary fat. Am J Clin Nutr 1997;66:97–103.
Danek GM, Valenti M, Baralle FE, Romano M. The A/G polymorphism in the –78 position of the apolipoprotein A-I promoter does not have a direct effect on transcriptional efficiency. Biochem Biophys Acta 1998;1398:67–74.
Xu CF, Angelico F, Del Ben M, Humphries SE. Role of genetic variation at the apo AI-CIII-AIV gene cluster in determining plasma apo A-I levels in boys and girls. Genet Epidemiol 1993;10:113–22.
Barre DE, Guerra R, Verstraete R, Wang Z, Grundy SM, Cohen JC. Genetic analysis of a polymorphism in the human apolipoprotein A-I gene promoter: effect on plasma HDL-cholesterol levels. J Lipid Res 1994;35:1.292-6.
Civeira F, Pocovi M, Cenarro A, Garcés C, Ordovás JM. Adenine for guanine substitution. 78 base pairs to the apolipoprotein (apo) A-I gene: relation with high density lipoprotein cholesterol and apo A-I concentrations. Clin Genet 1993;44:307–12.
Soriguer-Escofet F, Esteva I, Rojo-Martinez G, Ruiz de Adana S, Catala M, Merelo MJ, et al. Diabetes Group of the Andalusian Society of Endocrinology and Nutrition. Prevalence of latent autoimmune diabetes of adults (LADA) in Southern Spain. Diabetes Res Clin Pract 2002;56:213–20.
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215.
Queipo-Ortuno MI, Garcia-Ordonez MA, Colmenero JD, Morata P. Hydrogen peroxide improves the efficiency of a peripheral blood PCR assay for diagnosis of human brucellosis. Biotechniques 1999;27:248–50, 252.
Shoulders CC, Narcisi TM, Jarmuz A, Brett DJ, Bayliss JD, Scott J. Characterization of genetic markers in the 5' flanking region of the apo A1 gene. Hum Genet 1993;91:197–8.
Jeenah M, Kessling A, Miller N, Humphries S. G to A substitution in the promoter region of the apolipoprotein AI gene is associated with elevated serum apolipoprotein AI and high density lipoprotein cholesterol concentrations. Mol Biol Med 1990;7:233–41.
Dammerman M, Sandkuijl LA, Halaas JL, Chung W, Breslow JL. An apolipoprotein CIII haplotype protective against hypertriglyceridemia is specified by promoter and 3_UTR polymorphisms. Proc Natl Acad Sci USA 1993;90:4562–6.
Rathmann W, Funkhouser E, Dyer AR, Roseman JM. Relations of hyperuricemia with the various components of the insulin resistance syndrome in young black and white adults: the CARDIA study. Coronary Artery Risk Development in Young Adults. Ann Epidemiol 1998;8:250–61.
Perez-Ruiz F, Calabozo M, Erauskin GG, Ruibal A, Herrero-Beites AM. Renal underexcretion of uric acid is present in patients with apparent high urinary uric acid output. Arthritis Rheum 2002;47:610–13.
Tinahones FJ, Collantes E, C-Soriguer FJ, Gonzalez-Ruiz A, Pineda M, Anon J, et al. Increased VLDL levels and diminished renal excretion of uric acid in hyperuricaemic-hypertriglyceridaemic patients. Br J Rheumatol 1995;34:920–4.
Tinahones JF, Perez-Lindon G, C-Soriguer FJ, Pareja A, Sanchez-Guijo P, Collantes E. Dietary alterations in plasma very low density lipoprotein levels modify renal excretion of urates in hyperuricemic-hypertriglyceridemic patients. J Clin Endocrinol Metab 1997;82:1188–91.
Cardona F, Tinahones FJ, Collantes E, Escudero A, Garcia-Fuentes E, Soriguer FJ. The elevated prevalence of apolipoprotein E2 in patients with gout is associated with reduced renal excretion of urates. Rheumatology (Oxford) 2003;42:468–72.
Dallinga-Thie GM, Bu XD, van Linde-Sibenius Trip M, Rotter JI, Lusis AJ, de Bruin TW. 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 1996;37:136–47.
Meng QH, Pajukanta P, Valsta L, Aro A, Pietinen P, Tikkanen MJ. Influence of apolipoprotein A-1 promoter polymorphism on lipid levels and responses to dietary change in Finnish adults. J Intern Med 1997;241:373–8.
Wang XL, Badenhop R, Humphrey KE, Wilcken DE. C to T and/or G to A transitions are responsible for loss of a MspI restriction site at the 5'-end of the human apolipoprotein AI gene. Hum Genet 1995;95:473–4.
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Wang XL, Badenhop R, Humphrey KE, Wilcken DE. New MspI polymorphism at +83 bp of the human apolipoprotein AI gene: association with increased circulating high density lipoprotein cholesterol levels. Genet Epidemiol 1996;13:1–10.
Kessling AM, Horsthemke B, Humphries SE. A study of DNA polymorphisms around the human apolipoprotein AI gene in hyperlipidaemic and normal individuals. Clin Genet 1985;28:296–306.
Dallinga-Thie G, Linde-Sibenius M, Rotter J, Cantor RM, Xiang-dong B, Lusis AJ, et al. Complex genetic contribution of the Apo AI-CIII-AIV gene cluster to familial combined hyperlipidemia. identification of different susceptibility haplotypes. J Clin Invest 1997;99:953–61.(F Cardona, F J Tinahones,)
2 Servicio de Reumatologia, Hospital Reina Sofia, Cordoba, Spain
Background: Studies have shown that hyperuricaemia is independently related to the insulin resistance syndrome and that polymorphisms of the apolipoprotein AI-CIII-AIV cluster are also related to insulin resistance.
Objective: To study the prevalence of polymorphisms of the apolipoprotein AI-CIII-AIV cluster in persons with gout and to determine whether these polymorphisms contribute to the pathophysiology of gout or to altered lipid concentrations.
Methods: Plasma cholesterol, triglycerides, uric acid, VLDL, LDL, IDL, and HDL triglycerides, cholesterol, and the renal excretion of uric acid were measured in 68 patients with gout with gout and 165 healthy subjects. Polymorphisms were studied by amplification and RFLP in all subjects, using XmnI and MspI in the apolipoprotein AI gene and SstI in the apolipoprotein CIII gene.
Results: The A allele at position –75 bp in the apolipoprotein AI gene was more common in patients with gout than in controls (p = 0.01). Levels of cholesterol, triglycerides, uric acid, basal glycaemia, and HDL cholesterol were higher in the patients (p<0.001). In the patients there was also an interaction between mutations at the two polymorphic loci studied in the apolipoprotein AI gene (p = 0.04). An absence of the mutation at position –75 bp of the apolipoprotein AI gene resulted in increased plasma triglyceride levels.
Conclusions: Gouty patients have an altered allelic distribution in the apolipoprotein AI-CIII-AIV cluster, which could lead to changes in levels of lipoproteins. This is not caused by a single mutation but rather by a combination of different mutations.
Keywords: apolipoprotein AI-CIII-AIV cluster; gout; polymorphisms
Hyperuricaemia appears to be an independent risk factor for coronary heart disease.1–3 Although the reason for this is not well known, evidence suggests that hyperuricaemia forms part of the metabolic syndrome. A high concentration of uric acid is closely related to hyperinsulinaemia, glucose intolerance, hypertension, dyslipidaemia, very high levels of triglycerides, low levels of high density lipoprotein (HDL) cholesterol, and obesity (especially abdominal adiposity).4–12 Regarding the association that has already been demonstrated between hyperuricaemia and hyperlipidaemia, we have previously shown the existence of two groups of hyperuricaemic patients—those in whom the hyperuricaemia is associated with hyperlipidaemia, who could be considered to have the metabolic syndrome, and those having no association with any metabolic disorder.13 We have also reported that hyperuricaemic-hypertriglyceridaemic patients have high levels of very low density lipoprotein (VLDL) components, a reduced fractionated excretion of uric acid, and an increased ratio of CIII to CII apolipoproteins. Apolipoproteins therefore play an important role in the pathophysiology of the changes seen in hyperuricaemia.14
Apolipoproteins AI-CIII-AIV are lipid binding proteins that are involved in the transport of lipids in plasma. Defects or variations in these apolipoproteins are also associated with altered plasma concentrations of lipids and lipoproteins. The relation between variations in the apolipoprotein AI-CIII-AIV gene cluster and plasma lipids has long been recognised. Most studies have concentrated on the association between plasma triglycerides and variations in the gene cluster.15 Upregulation of apolipoprotein CIII in transgenic mice induces hypertriglyceridaemia because this apoprotein is involved in VLDL clearance.16 The apoprotein CIII gene is closely linked to the apolipoprotein AI and AIV genes on chromosome 11, forming a cluster. Study of polymorphisms in this cluster has shown that the S2 allele of the polymorphism is related to hypertriglyceridaemia and an increased risk for coronary heart disease.17,18 López-Miranda et al found that carriers of the S2 allele had reduced concentrations of LDL cholesterol when given a diet rich in monounsaturated fats.19 Furthermore, the change of a G for an A at position –75 base pairs (bp) in the promoter region of the apolipoprotein AI gene, digested by the restriction enzyme MspI, induces increased transcription of this gene and increased serum levels of apolipoprotein AI.20 Individuals with this mutation also have higher levels of HDL cholesterol. Although not yet demonstrated, it would appear that this can be explained by linkage disequilibrium between the A allele and another functional mutation in the gene. There is another mutation in the 5' region near the apolipoprotein AI gene –2500 bp upstream from the restriction start site, which is the marker for combined family hyperlipidaemia or hypertriglyceridaemia.21–23 These data show that the cluster of apolipoprotein AI-CIII-AIV genes is involved in hypertriglyceridaemia, and may also be involved in predisposition to atherosclerosis.
We studied the prevalence of polymorphisms of the apolipoprotein AI-CIII-AIV cluster in persons with gout and determined whether these polymorphisms contribute to the pathophysiology of hyperuricaemia or to altered lipid levels in these individuals.
Subjects
The study was undertaken in 68 men with gout attending the rheumatology service at the Hospital Reina Sofía in Cordoba, Spain. None was receiving lipid lowering or urate lowering treatment. Height and weight were measured and the body mass index (BMI) calculated.
Patients with secondary hyperuricaemia (renal failure or use of diuretics) were excluded from the study.
A control group was composed of 165 healthy persons with no history of gout and with cholesterol and triglyceride concentrations lower than 5.2 and 2.3 mmol/l, respectively, selected from a local cross sectional study24 and adjusted for age.
Procedures
DNA analysis
DNA was isolated from venous blood by the "salting out" method of Miller et al,25 modified by Queipo-Ortu?o et al,26 and amplified by polymerase chain reaction in a thermocycler (mastercycler Eppendorff). Primers for the XmnI locus (at position –2500 bp upstream of the transcription start site of the apolipoproteins AI) were those described by Shoulders et al,27 while those of Jeenah et al were used for the –75 bp locus of the apolipoprotein AI gene (determined by MspI),28 and those of Dammerman et al for the SstI locus of the apolipoprotein CIII gene.29
Laboratory analysis
Venous blood samples, collected after a 12 hour fast, were ultracentrifuged at 2000xg for 15 minutes for measurement of lipids and lipoproteins. Plasma levels of glucose, cholesterol, triglycerides, creatinine, HDL cholesterol, low density lipoprotein (LDL) cholesterol, and uric acid were measured in an autoanalyser using calorimetric assays (Ecoline 25, Diagnostica Merck). Calculations were made of uric acid clearance and the 24 hour uric acid urinary excretory fraction.
Lipoproteins were separated from serum by ultracentrifugation as follows: first, the VLDL fraction was separated from the serum by flotation at 125 000xg for 18 hours at 7°C in a 45° rotor (Beckman TLA 100.3) After separation of the VLDL, the density of the infranatant was adjusted to 1.30 g/ml by direct addition of potassium bromide and saccharose, separating the rest of the lipoproteins by density gradient centrifugation at 208 000xg for 22 hours at 7°C in a swinging bucket rotor (Beckman SW 40Ti). To visualise the position of the lipoprotein bands a small amount (25 μl) of saturated Coomassie blue was added. The concentrations of cholesterol and triglycerides were measured in each fraction as above. The amount of HDL cholesterol was also measured by the alternative method of precipitation with phosphotungstic acid in patients and controls. The LDL cholesterol was calculated from the Friedewald equation.
Statistical analysis
Data are expressed as mean (SD). Comparison between groups was made using Student’s t test for independent variables or the Mann–Whitney U test, depending on the normality of the distribution. Differences in the genotype distribution of the apolipoprotein AI-CIII-AIV cluster were studied by 2 tests. Statistical analyses were carried out with SPSS 6.0 for Windows and probability (p) values <0.05 were considered significant.
The clinical and laboratory data for the patients and controls are summarised in table 1. Plasma concentrations of cholesterol, triglycerides, and uric acid were higher in patients with gout than in the controls: (mean (SD) 5.48 (1.04) v 4.49 (0.45); 2.79 (2.51) v 1.01 0.45); and 0.46 (0.10) v 0.31 (0.07) mmol/l, respectively; p<0.001. The patients also had higher plasma HDL cholesterol levels (1.22 (0.32) v 1.08 (0.24) mmol/l; p<0.001) and a higher BMI (30.2 (3.8) v 27.3 (3.7) kg/m2; p<0.001) than the controls (table 1).
Frequency distribution of the polymorphisms of the apolipoprotein AI-CIII-AIV cluster
The frequency of the A allele—that is, the presence of the mutation in both homozygotes and heterozygotes—was higher in patients than in controls (p = 0.01). The distribution of the different genotypes of this polymorphism is shown in table 2. The unusual alleles in homozygotes located –2500 bp upstream of the transcription start site of the apolipoprotein AI gene (determined by XmnI) and of exon 4 of the apolipoprotein CIII gene (determined by SstI) were also more frequent in patients than controls, though the difference was not significant.
Influence of the polymorphisms of the apolipoprotein AI-CIII-AIV cluster on biological variables
The effect of these polymorphisms on the study variables differed. The presence of the X2 allele in the controls was not related to any increase in the variables studied, whereas its presence in the patients was related to an increased plasma concentration of triglycerides (2.31 (1.68) v 3.82 (3.76) mmol/l; p = 0.03) (table 3). The other polymorphisms studied in the apolipoprotein AI-CIII-AIV cluster were not related significantly to any differences in the distribution of the study variables (data not shown).
The content of VLDL triglycerides was greater in those patients with the X2 allele than in non-carriers, adjusted for age and BMI (1.77 (1.96) v 1.05 (0.71) mmol/l; p = 0.05) (table 3). The levels of IDL and LDL triglycerides were also higher in carriers of the mutated allele, though the difference was not statistically significant. There were no significant differences in any of the other lipoprotein fractions studied (table 3), or in the distribution of the biological variables in either of the other two polymorphisms of the apolipoprotein AI and CIII cluster (data not shown).
The combined distribution of the polymorphisms of the apolipoprotein AI-CIII-AIV cluster differed between the patients and the controls; 63.5% of the patients had some mutation at the two polymorphic sites of the apolipoprotein AI gene studied, compared with 41.7% of the controls (p = 0.007) (table 4). The presence of at least one unusual allele was related to significant differences in the lipid content of the lipoprotein fractions (table 5). VLDL cholesterol was higher in patients with at least one mutation in any of the polymorphisms of the cluster than in patients with no mutation (p = 0.044). The triglyceride content was greater in carriers of some mutation in the cluster than in persons with no mutation (p = 0.014). The IDL cholesterol was higher in carriers of some mutation than in non-carriers (p = 0.021). HDL cholesterol was also higher in carriers, but the difference was not significant.
Hyperuricaemia is a risk factor for coronary heart disease.30 The relation between hyperuricaemia-hyperlipidaemia and renal excretion of urates is known—individuals with hyperuricaemia-hyperlipidaemia have reduced renal excretion of urates compared with those who have only isolated hyperuricaemia; and the latter have also been reported to have low renal excretion of urates.31,32 In patients with hyperuricaemia and increased VLDL there is a close relation between the VLDL levels and renal excretion of urates.32,33 This relation appears to be mediated through the high prevalence of the E2 isoform of apolipoprotein E.34
Recent studies have related polymorphisms of the cluster to variations in plasma lipid levels. Variations in these genes contribute to combined family hyperlipidaemia, modifying plasma concentrations of cholesterol and triglycerides, as well as those of apolipoprotein B and apolipoprotein CIII.35
Patients with gout have a greater prevalence of mutated genotypes at polymorphic sites of the apolipoprotein AI gene; in our study, 18.8% had AA at position –75 bp from the transcription start site v 6.7% in controls (p = 0.01). This high prevalence may be responsible for the high levels of HDL cholesterol seen in patients with gout, as has been described for other disease groups.36–39
We also found a greater frequency of the mutated allele at position –2500 bp in the apolipoprotein AI gene, although the difference was not significant. We showed that 63.5% of persons with gout have some mutation at the two polymorphic sites of the apolipoprotein AI gene, compared with 36.7% who do not have these alleles (p = 0.007). These patients with a mutated allele at position –2500 bp upstream of the apolipoprotein AI gene had higher plasma and VLDL triglyceride levels than those without the mutation (p = 0.05), corrected for BMI (patients with the mutation had a lower BMI than those without the mutation). This allele has been associated with hypertriglyceridaemia in patients with combined family hyperlipidaemia.40,41 In our group of patients there seemed to be a synergic effect of the two polymorphic sites in the influence on plasma triglycerides. Patients with gout who had some mutation at the three polymorphic sites studied had higher contents of VLDL, intermediate density lipoprotein (IDL), HDL cholesterol, and VLDL triglycerides. Thus their metabolism of lipoproteins seems to be altered, thereby contributing to the expression of hypertriglyceridaemia. The lipoprotein phenotype characteristic of the insulin resistance syndrome was more prevalent in the gouty subjects with a mutation in the cluster despite their BMI being no greater than in subjects without the mutation.
We conclude that the high prevalence of mutations in the apolipoprotein AI-CIII-AIV cluster in patients with gout partly explains their lipoprotein phenotype and that, jointly, these mutations exert more influence on the polymorphic sites of the apolipoprotein AI gene than the apolipoprotein CIII gene.
ACKNOWLEDGEMENTS
This study was supported by grants from "Red de metabolismo y nutricion Fis C03/08" and a predoctoral grant from the Fundación Hospital Carlos Haya. We want to thank Pablo Rodriguez and Isabel Recio for excellent laboratory technical assistance, and gratefully acknowledge Ian Johnstone for his expertise in preparing the manuscript.
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