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Protease inhibitor-associated dyslipidemia in HIV-infected patients is strongly influenced by the APOA5-1131T [right arrow] C gene variation.

HIV-infected individuals have higher rates of subclinical atherosclerosis than the age-adjusted general population (1), and the incidence of cardiovascular events is directly related to the years of exposure to antiretroviral therapy (2, 3). The metabolic abnormalities associated with antiretroviral therapy (4), chronic inflammatory status (5), and the HIV infection itself (6) have been postulated as possible causes of this increased susceptibility to cardiovascular disease. Hypercholesterolemia and hypertriglyceridemia, which are well-established, independent, cardiovascular disease risk factors, are associated with the use of protease inhibitors, especially in patients undergoing ritonavir or ritonavir-boosted antiretroviral treatment. Despite similar antiretroviral treatment and demographic characteristics, however, not all HIV-infected patients develop these metabolic and cardiovascular complications. Hyperlipidemia, and more importantly, hypertriglyceridemia in HIV patients is highly influenced by genetic variability (7) and, among the candidate genes, the newly identified apolipoprotein A-V (APOA5) [1] has emerged as probably the most potent modulator of triglyceride (TG) [2] metabolism. The role of APOA5 in regulating TG metabolism has been convincingly demonstrated in animal models (8) and in a large number of association studies (9), in which the -1131T [right arrow] C is the commonly used variant. APOA5 is expressed mainly in the liver and distributed predominantly on TG-rich lipoproteins such as chylomicrons and VLDL, and also on HDL. In human plasma, apo A-V is found at lower concentrations than other apolipoproteins. Its exact function has not been completely elucidated, but the available data indicate that it modulates TG metabolism by controlling production of VLDL and catabolism of the lipolysis of TG-rich lipoproteins (10). The latter role has been confirmed not only in vitro but also in patients who develop severe hypertriglyceridemia attributable to apo A-V deficiency (11).

Because protease inhibitor (PI)-associated dyslipidemia is caused by increases in VLDL production (12) and by impaired lipolysis (13), we hypothesized that, in HIV-infected individuals, the APOA5 gene could be an important indicator of predisposition to PI-related deterioration of the lipid profile. Hence, we analyzed lipid changes in HIV patients segregated with respect to treatment strategy and APOA5 genotype.

Materials and Methods


A total of 229 HIV-infected patients attending our outpatient clinic gave written informed consent to participate in the study. The present study is part of a longitudinal project in which atherosclerosis in HIV-infected patients is being assessed in a cohort of patients who are followed regularly in our outpatient clinic. The overall characteristics of these patients have been described previously (14,15). Time 0 of the study corresponds to commencement of therapy with PIs (n = 148) or without PIs (n = 81). Subsequent time-points are at 1, 2, 3, 4, and 5 years of treatment follow-up. Clinical and biochemical data and the APOA5-1131T [right arrow] C genotype have been measured for all participants. Exclusion criteria were age <18 years and AIDS-related opportunistic disease when the study began. The Ethics and Investigation Committee of our Hospital approved the study.


We used standard laboratory methods to measure total cholesterol, HDL cholesterol, and TGs.


We measured serum high-sensitive C-reactive protein (hsCRP) concentrations with a turbidimetric immunoassay (Biokit), according to the manufacturer's instructions.

APOAS -1131T [right arrow] C GENOTYPE ANALYSES

Genomic DNA was obtained from leukocytes and extracted with calibrated methods. The -1131T [right arrow] C variation in the APOA5 gene was detected with the oligonucleotide primers AV-1-5'-GAT TGA TTC AAG ATG CAT TTA GGA C-3' and AV-2-5'000 CAG GAA CTG GAG CGA AAT T-3' to amplify a 187op segment, and AV-2 primer forced a site for Mse I (New England Biolabs) enzymatic restriction. PCR was performed as described previously (8).


We used a multivariate analysis on patients with complete data available [age, body mass index (BMI), sex, and lipid data at all time points] being entered into the analysis. To improve the statistical power of our dataset, we performed ANOVA analyses of data from every single time-point, used lipid values as a dependent variable, and normalized for confounding factors such as sex, age, BMI, and lipodystrophy. We also performed multivariate analyses of repeated measures, which confirmed the trends but did not reach statistical significance. For TG and hsCRP, calculations were performed on log-transformed values, although nontransformed concentrations are shown in the Tables and Figs.

We analyzed deviation from Hardy Weinberg equilibrium with the [chi square] goodness-of-fit test. Results are conveyed as mean (SD). Statistical significance was accepted at a value of P <0.05.


The data from a total of 229 HIV-infected patients were segregated according to APOA5 -1131T [right arrow] C genotype and treatment scheme. For statistical purposes, the single patient homozygous for the C allele was pooled with those patients who were T/C heterozygote. For lipid analyses, patients undergoing the PI regimen (n = 148) and those not receiving PIs (n = 81) were studied separately. The frequency of the C allele was 0.08, which is similar to that found in the Spanish general population (0.07) (16). Allelic distribution was in Hardy-Weinberg equilibrium.


The group of patients with the wild-type genotype (TT) and carriers of the rare variant (TC and CC) were comparable at baseline with respect to age, sex, immunologic status, hsCRP, total cholesterol, HDL cholesterol, and TGs (Table 1). Only BMI was considerably higher in the carriers of the wild-type allele.


Because hyperlipidemia is strongly associated with the PI regimen, we focused on the subgroup of 148 patients receiving PI as a component of their antiretroviral therapy, and we analyzed their lipid profile changes over the 5-year follow-up period. The 2 genotype groups were also comparable at baseline (pretreatment), including the percentage of patients receiving ritonavir (Table 1).

Carriers of the C allele had consistently higher TG concentrations than noncarriers at 1 year [2.11 (1.62) vs 3.71 (4.27) mmol/L; P = 0.009], 2 years [2.48 (2.09) vs 4.02 (4.05) mmol/L; P = 0.050], 3 years [2.32 (1.71) vs 4.13 (4.26) mmol/L, P = 0.013], 4 years [2.90 (2.95) vs 5.35 (7.12) mmol/L; P not significant], and 5 years [4.25 (5.58) vs 9.23 (9.63) mmol/L; P not significant], after adjustment of the data for age, sex, BMI, and the presence of lipodystrophy (Fig. 1).

Results were similar for total cholesterol. Carriers of the C allele had higher plasma cholesterol concentrations at 1 year [4.93 (1.31) vs 5.87 (1.66) mmol/L; P = 0.006], 2 years [5.03 (1.12) vs 6.42 (2.48) mmol/L; P = 0.001], 3 years [5.11 (1.17) vs 6.38 (2.43) mmol/L; P = 0.009], 4 years [5.49 (1.71) vs 6.78 (3.03) mmol/L; P = not significant], and 5 years [5.56 (1.75) vs 7.90 (3.60) mmol/L; P = not significant] (Fig. 2). HDL cholesterol concentrations showed a tendency toward decrease in carriers of the C allele and increase in patients with the wild-type alleles, but these differences did not reach statistical significance (data not shown).

The total cholesterol/HDL cholesterol ratio, which was 78% higher in carriers of the C allele than in carriers of the wild-type allele (Fig. 3), indicated that these lipid changes increased the risk of atherogenesis.


To investigate whether the effect of APOA5 on the lipid profile in HIV patients was influenced by treatment with PIs or whether the effect was more generalized, we separately evaluated the 81 patients who were not receiving treatment with PIs. At baseline, the 2 genotype groups were comparable (Table 1). There were no differences between genotypes with respect to total cholesterol, TGs, HDL cholesterol, or the total cholesterol/HDL cholesterol ratio over the 5-year follow-up period (Figs. 1-3).


Our results show that HIV-infected patients with the APOA5-1131C allele are predisposed to severe hyperlipidemia related to treatment with PIs, i.e., the adverse effects of PI appear to be exacerbated in patients with the C allele on the APOA5 gene.


The PIs used in combined therapies can produce major fat redistribution, hyperlipidemia, and insulin resistance. These effects can be mitigated by replacing PIs with other antiretroviral drugs (17). That these abnormalities do not develop in all patients on PI regimens suggests the involvement of genetic or environmental predisposing factors. We focused on the APOA5 gene because it is probably the strongest genetic determinant of plasma TG (9) identified to date, and few data on the influence of APOA5 on PI-induced hyperlipidemia are available.

Among the 229 HIV-patients we followed for a period of 5 years, those receiving the PI regimen tended, as expected, to have higher concentrations of total cholesterol and TGs during the treatment period. Despite similar baseline lipid values among all patients, however, the individuals carrying the -1131C variant of the APOA5 gene and undergoing treatment with PIs had the highest values at all of the follow-up time-points. Conversely, carriers of the -1131C variant not receiving PI treatment did not have such strongly increased concentrations of these lipids. This observation is in accordance with our previous studies demonstrating that the magnitude of the effect of the APOA5 gene is more pronounced when conditions are more metabolically challenging (16).


Increased cholesterol concentrations in carriers of the -1131C allele could also be the result of increased cholesterol delivery by the TG-rich lipoproteins, a well-described secondary feature of VLDL overproduction. Although detailed lipoprotein subfractionation was not available in the present group of patients, it is clear that increased VLDL synthesis, decreased catabolism, or a combination of these processes can lead to PI-induced hyperlipidemia. It is also clear that these metabolic perturbations are characterized essentially by an increasing TG component with a concomitant increase in the cholesterol concentration in these lipoproteins.


In our study sample, carriers of the APOA5 variant allele had higher rates of lipodystrophy. The APOA5 gene may somehow predispose individuals to this lipid and fatty-tissue redistribution, but this hypothesis is not supported by our finding that the frequency of lipodystrophy among APOA5 genotypes in the non-PI group did not differ from the frequency in the overall patient group. Conversely, after adjustment for the presence of lipodystrophy, all of the observed differences with respect to lipid concentrations remained considerable, indicating that the hyperlipemic effect was truly associated with the APOA5 gene and was not influenced by the presence of lipodystrophy.


A mechanistic approach to this observation could be that, although the exact function in vivo of apo A-V is not known, in vitro studies suggest that it acts by decreasing the assembly of the VLDL particle (18) and its secretion and by stimulating lipolysis in the circulation (10). Conversely, PI-induced hyperlipidemia has been shown to be caused by an increase in VLDL production (12) as well as by impaired lipolysis (13) as a consequence, in part, of a direct interaction between the PI and the sterol regulatory element-binding proteins (SREBP)1 and 2, leading to an accumulation of SREBPs in the nucleus, which stimulates lipid synthesis (19). Because APOA5 expression is down-regulated by SREBP1c (20), we assume that apo A-V plays a determining role in PI-induced hyperlipidemia. Whether the -1131C allele is a functional variant or is acting as a marker of a functional variant elsewhere in the gene is not clear (21).


Patients who are receiving PI-treatment and who carry the -1131C allele present with a lipoprotein profile that deteriorates rapidly because of increasing of total cholesterol and TGs and a decrease in the HDL fraction, with the atherogenic total/HDL cholesterol ratio reaching the highest quintile after 3 years in carriers of the mutant C allele (22). This scenario does not occur in patients who are receiving the same treatment but who carry the wild-type gene or who are not receiving PI therapy.

One of the limitations of our study is that there were fewer patients available to follow up at years 4 and 5 than for the first 3 years because 4 to 5 years ago there was less concern regarding lipid alterations in individuals with HIV-AIDS, so the percentage of individuals with HIV-AIDS being treated for hyperlipidemia was considerably lower in the first 2 years of recruitment into the present study. We suspect that the patients who were recruited were those with more evident dyslipidemia, which might explain the greater increase in plasma TG concentrations in year 5. It is important to note, however, that all the main conclusions of the study were drawn from the data obtained during 1-3 years of follow-up. Data from 4-5 years of follow-up were included for completeness, and to indicate that the trends toward increased lipids observed in the first 3 years continued in the same direction over the subsequent years of follow-up in those patients, for whom detailed lipid datasets were available.

In conclusion, our results indicate that variability at the APOA5 gene variation predisposes HIV patients, particularly those treated with PIs, to severe hyperlipidemia. Although these results must be confirmed in future studies, they suggest the possibility of using the APOA5 gene as a marker of predisposition to severe hyperlipidemia as a consequence of treatment with PIs. In HIV-positive individuals found to carry this variation, treatment alternatives that exclude PI may need to be considered.

This study was supported by grants from the Instituto de Salud Carlos III, Red de Centros de Metabolismo y Nutricion (C03/08), genetic hyperlipidemias (G03/181), Mediterranean diet (G03 / 140), and SAF-2002-02781 (Madrid, Spain). Dr. Blai Coll is in receipt of a career development award from the Instituto de Salud Carlos III (Madrid, Spain). Montse Guardiola is a recipient of a predoctoral fellowship at the Spanish Ministry of Science and Technology (BES-2003-1090).


(1.) Hsue PY, Lo JC, Franklin A, Bolger AF, Martin JN, Deeks SG, et al. Progression of atherosclerosis as assessed by carotid intimamedia thickness in patients with HIV infection. Circulation 2004; 109:1603-8.

(2.) Mary-Krause M, Cotte L, Simon A, Partisani M, Costagliola D. Increased risk of myocardial infarction with duration of protease inhibitor therapy in HIV-infected men. Aids 2003;17:2479-86.

(3.) Frus-Moller N, Sabin CA, Weber R, d'Arminio Monforte A, El-Sadr WM, Reiss P, et al. Combination antiretroviral therapy and the risk of myocardial infarction. N Engl J Med 2003;349:1993-2003.

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Institut de Recerca en Ciencies de la Salut, Hospital Universitari de Sant Joan de Reus, Universitat Rovira i Virgili, Reus, Spain

[1] Human gene: APOA5, apolipoprotein A-V.

[2] Nonstandard abbreviations: TG, triglyceride; PI, protease inhibitor; HsCRP, high-sensitive C-reactive protein; SREBP, sterol regulatory element-binding proteins; BMI, body mass index.

* Address correspondence to this author at: Unitat de Recerca de Lipids i Arteriosclerosi, Facultat de Medicina, Universitat Rovira i VirgIIi, Sant Llorenc, 21, 43201 Reus, Spain. Fax 34-977-75-9322; e-mail

([dagger]) These authors contributed in equal measure to the present study.

Received March 1, 2006; accepted July 18, 2006.

Previously published online at DOI: 10.1373/clinchem.2006.069583
Table 1. Baseline selected epidemiological, immunological, and
inflammatory characteristics of study participants segregated with
respect to APOA5 genotype.

 All APOA5
 participants APOA5 (TT) (TC+CC)
 n = 229 n = 191 n = 38

Age, years 38.6 (6.8) 38.5 (6.7) 40.1 (9.2)
Gender, % male 158 (62.9) 135 (69.9) 25 (65.7)
BMI, kg/[m.sup.2] 23 (3.1) 23.2 (3) 21.6 (2.9)
CRP, mg/L 4 (5.6) 4 (5.7) 4.42 (5)
Total cholesterol, mmol/L 4.60 (1.20) 4.60 (1.21) 4.58 (1.22)
Triglyceride, mmol/L 2.00 (2.05) 1.99 (2.15) 2.05 (1.47)
HDL cholesterol, mmol/L 1.1 (0.4) 1.1 (0.4) 1.2 (0.2)
Nadir CD4, cell/[mm.sup.3] 344 (286) 343 (302) 449 (281)
CD4 <200 [mm.sup.3], % 65 (28.1) 56 (29) 9 (23.6)
HIV viral load, copies/mL 254 377 302 331 294 004
 (793 524) (938 393) (508 390)
Lipodystrophy, % 20.9 (24.1) 37 (22.7) 10 (30.3)
Ritonavir boosted, %

 P (TT) (TC+CC) P
 n = 128 n = 20

Age, years NS (a) 39.3 (7.4) 39.7 (6) NS
Gender, % male NS 92 (71.8) 16 (80) NS
BMI, kg/[m.sup.2] 0.03 23 (2.8) 22.1 (3.4) NS
CRP, mg/L NS 4.2 (6.4) 5.9 (5.5) NS
Total cholesterol, mmol/L NS 4.63 (1.31) 5.06 (1.02) NS
Triglyceride, mmol/L NS 2.14 (2.41) 2.17 (1.84) NS
HDL cholesterol, mmol/L NS 1.1 (0.5) 1.3 (0.2) NS
Nadir CD4, cell/[mm.sup.3] NS 327 (321) 300 (309) NS
CD4 <200 [mm.sup.3], % NS 44 (34.3) 6 (30) NS
HIV viral load, copies/mL NS 351 107 NA NS

Lipodystrophy, % NS 28 (25.2) 9 (47.4) 0.04
Ritonavir boosted, % 33 (25.7) 7 (35) NS

 (TT) (TC+CC) P
 n = 63 n = 18

Age, years 37.8 (5.4) 35 (8.7) NS
Gender, % male 32 (51) 6 (33) NS
BMI, kg/[m.sup.2] 23.6 (3.5) 21.5 (2.8) 0.05
CRP, mg/L 3.3 (3.4) 2.6 (3.8) NS
Total cholesterol, mmol/L 4.53 (0.98) 3.98 (1.23) 0.04
Triglyceride, mmol/L 1.67 (1.41) 1.87 (0.75) NS
HDL cholesterol, mmol/L 1.1 (0.3) 1.1 (0.2) NS
Nadir CD4, cell/[mm.sup.3] 340 (190) 453 (285) NS
CD4 <200 [mm.sup.3], % 11 (16.9) 2 (11.1) NS
HIV viral load, copies/mL 144 467 159 354 NS
 (206 574) (248 845)
Lipodystrophy, % 8 (12.3) 1 (5.5) NS
Ritonavir boosted, %

Values are expressed as mean (SD). NS, not significant; NA, not
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Title Annotation:Lipids, Lipoproteins, and Cardiovascular Risk Factors
Author:Guardiola, Montse; Ferre, Raimon; Salazar, Juliana; Alonso-Villaverde, Carlos; Coll, Blai; Parra, Sa
Publication:Clinical Chemistry
Date:Oct 1, 2006
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