Effects of APOE, APOB and LDLR variants on serum lipids and lack of association with xanthelasma in individuals from Southeastern Brazil

HUMAN AND MEDICAL GENETICS

RESEARCH ARTICLE

Effects of APOE, APOB and LDLR variants on serum lipids and lack of association with xanthelasma in individuals from Southeastern Brazil

Marcelo A. Nakazone^I; Miguel A. De Marchi^I; Marcela A.S. Pinhel^I; Carolina F.D.C. Barros^I; Maysa A.F. Júlio^I; Anielli Pinheiro^I; Simone S. Arazi^II; Júlia K. Hotta^III; Mário H. Hirata^II; Rosario D.C. Hirata^II; José E. dos Santos^III; Dorotéia R.S. Souza^I

^IDepartamento de Biologia Molecular, Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, SP, Brazil

^IIDepartamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil

^IIIDepartamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

^{Send correspondence to}

ABSTRACT

Xanthelasma might be a clinical manifestation of dyslipidemia, a recognized risk factor for coronary artery disease. We investigated the association of apolipoprotein E (APOE HhaI), apolipoprotein B (APOB XbaI and Ins/Del) and LDL receptor (LDLR AvaII and HincII) gene polymorphisms with lipid profiles in 100 Brazilians with xanthelasma and 100 controls. Allele frequencies were similar in both groups. APOE, APOB and LDLR genotypes were not correlated with differences in the serum lipid profile. In individuals with xanthelasma, the APOB D allele was associated with less chance of having increased LDL-cholesterol (O.R. = 0.16, CI95% = 0.03-0.94, p = 0.042). In the control group, the APOB X+ allele was associated with less chance of having both increased total cholesterol (O.R. = 0.16, CI95% = 0.03-0.78, p = 0.023) and increased LDL-cholesterol (O.R. = 0.10, CI95% = 0.02-0.60, p = 0.012). Moreover, there was a significantly higher frequency of control individuals (68%) with elevated serum triglyceride levels, compared to patients (48%, p = 0.008). On the other hand, triglyceride levels in controls also seemed to be influenced by all other gene polymorphisms studied, an effect that might be enhanced by environmental factors.

Key words: apolipoprotein B, apolipoprotein E, LDL receptor, gene polymorphisms, xanthelasma.

Introduction

Xanthelasma is the most common form of lipid deposit on the skin, and appears as yellowish plaques on the eyelid or periorbital skin (Bergman, 1994). These have been considered as clinicals sign of both dyslipidemia and advanced atherosclerotic processes, implicated in the pathogenesis of coronary artery disease (CAD) (Ribera et al., 1995), although they have also been found in non-dyslipidemic individuals, thereby suggesting that other factors might be responsible for the formation of these atherogenic manifestations (Tursen et al., 2006).

Variants in the genes encoding apolipoproteins E (APOE) and B (APOB), as well as the low-density lipoprotein receptor (LDLR), have been associated with hypercholesterolemia and the increased risk of CAD in several populations (Hansen et al., 1993; Series et al., 1993; Ribera et al., 1995; Salazar et al. 1999; 2000; Cavalli et al., 2000; Pongrapeeporn et al., 2000; Guo et al., 2002; Hirashiki et al., 2003; Rios et al., 2003; Souza et al., 2007). Xanthelasma in association with cardiovascular disease has been reported in individuals with familial hypercholesterolemia and carrying LDLR mutations (Norman et al., 1999), and have also been associated with the presence of the APOE*4 allele (Ribera et al., 1995).

With the aim of elucidating the contribution of common polymorphisms from those genes involved in lipid metabolism to the development of xanthelasma, we evaluated whether APOE (HhaI), APOB (XbaI and Ins/Del) and LDLR (AvaII and HincII) gene polymorphisms were associated with the lipid profile encountered in Brazilian individuals with xanthelasma.

Material and Methods

Subjects

We investigated 100 unrelated individuals of European descent from southeastern Brazil presenting xanthelasma (37 men and 63 women, between the ages of 32 and 60 years). They were selected by a medical dermatologist in a private dermatology clinic, during the period from April, 2002 to March, 2005. Individuals with liver, renal or thyroid diseases, hypertension, diabetes mellitus, family dyslipidemia, and who had been using lipid-lowering drugs during the prior six months, at least, were precluded. The control group consisted of 100 individuals chosen from the general population (36 men and 64 women, aged from 32 to 57 years), and without any clinical signs of xanthomatosis or any other lipoid metabolic disorder. This study was approved by the Ethics Research Committee of the Faculdade de Medicina de São José do Rio Preto, SP, Brazil (protocol 2277/2002). Furthermore, all the participants signed informative forms, thereby indicating their consent.

Histopathology

The diagnosis of xanthelasma in all patients was confirmed through histopathological methods. Periorbital skin lesions were surgically excised the tissues being immediately immersed in Carnoy's methanolic fixative (60% methanol, 30% chloroform, and 10% glacial acetic acid) containing 25 µmoles/L of butylated hydroxytoluene and 50 pmoles/L of diethylenetriamine pentaacetic acid, to inhibit any ex vivo oxidative modification. The fixed samples were embedded in paraffin according to Sugiyama et al. (1992). Xanthelasma histological diagnosis was based on Breier et al. (2002) criteria.

Serum lipid profile

The lipid profile in serum was determined from peripheral blood samples drawn after a 12 hours' fast. The serum concentrations of triglycerides (TG) and total cholesterol (TC) were defined by enzymatic colorimetric methods (Bucolo and David, 1973; Allain et al., 1974). High-density lipoprotein cholesterol (HDL-cholesterol) was determined by precipitation with dextran-magnesium chloride. Low-density lipoprotein (LDL-cholesterol) and very low-density lipoprotein cholesterol (VLDL-cholesterol) were calculated by the Friedewald formula for TG levels below 400 mg/dL (Friedewald et al., 1972). TC/HDL-cholesterol, LDL-cholesterol/HDL-cholesterol and TG/HDL-cholesterol ratios were calculated (Da Luz et al., 2005). Reference values were those recommended by the III Brazilian Guidelines on Dyslipidemias and Guideline of Atherosclerosis Prevention from Atherosclerosis Department of Sociedade Brasileira de Cardiologia (Santos and Sociedade Brasileira de Cardiologia, 2001).

Genomic DNA analysis

Genomic DNA was extracted from peripheral blood leukocytes. APOE [APOE*2 (Cys176Arg, rs7412), APOE*4 (Cys130Arg, rs429358)], APOB [XbaI (Thr2515Thr, rs693)], LDLR [AvaII (Val653Val, rs5925) and HincII (Asn591Asn, rs688)] gene polymorphisms were analyzed through the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The APOB [Ins/Del (rs17240441)] variant was determined by PCR. Amplification was carried out in an Eppendorf-Mastercycler thermal cycler (Eppendorf HQ, Hamburg, Germany). Primers and PCR conditions had been previously described (Hixson and Vernier, 1990; Renges et al., 1991; Boerwinkle et al., 1990; Hobbs et al., 1992). PCR products were analyzed by 1.5% agarose gel electrophoresis after ethidium bromide staining. These were then digested with the endonucleases (Amersham Pharmacia Biotech of Brazil) HhaI and XbaI for APOE and APOB single nucleotide polymorphisms (SNPs), respectively, and AvaII and HincII for LDLR SNPs, according to manufacturer's instructions. Fragments were identified by electrophoresis on 2% agarose gels (APOB XbaI) and 6% polyacrylamide gels (APOB Ins/Del, APOE HhaI, LDLR AvaII and HincII). Homozygous samples for restriction sites were used as positive control, and 10% of the analyses were repeated randomly.

Statistical analysis

Categorical variables were compared by chi-squared or Fisher Exact tests. Continuous variables were compared through Mann-Whitney tests. Differences in genotype frequency distribution from Hardy-Weinberg ratios were verified by chi-squared tests. Genotype frequencies were estimated by means of the Expectation-Maximization algorithm using the Arlequin program. Relationships between xanthomatosis and genotypes and other variables were evaluated by univariate logistic regression and multivariate logistic regression analyses with stepwise criteria. The computer package SAS System 6.12 for Windows (SAS Institute Inc., Cary, NC, USA) was used throughout data analyses. Significance was defined as p < 0.05, after application of the Bonferroni correction.

Results

Biopsy of periorbital skin lesions from all the patients showed a localized collection of histiocytes with a foamy vacuolated cytoplasm, few lymphocytes and neutrophils in the dermis, the absence of fibrosis, and the presence of giant Touton cells (data not shown).

Table 1 shows demographic, biochemical and genotypic data from the study and control groups. Tested variables revealed no diversity between individuals with xanthelasma and controls. Allelic frequencies for APOE, APOB and LDLR polymorphisms were similar in both groups, and all the five polymorphisms exhibited the distribution patterns predicted by Hardy-Weinberg equilibrium (HWE), except APOB XbaI for patients (x²₍₁₎ = 3.97; 0.02 < p < 0.05).

No relationships were detected between gene polymorphisms and serum lipids in either of the two groups (Tables 2 and 3). In the control group, an increased TG/HDL-cholesterol ratio was observed in LDLR H+H+ genotype carriers (p = 0.036) when compared to non-carriers (data not shown).

Univariate logistic regression analysis showed that individuals with xanthelasma stand less chance of having increased TG (p = 0.014) and VLDL-cholesterol (p = 0.014; Table 4), and that individuals with lower TG (< 150 mg/dL) present an increased risk for xanthelasma (O.R. = 2.30, CI95% = 1.30-4.09, p = 0.005) (data not shown). Nevertheless, no relationships between gene polymorphisms and xanthelasma were found (Table 4).

Multivariate logistic regression analysis indicated that the presence of APOB X+ allele was associated with less chance of a rise in TC (> 200 mg/dL) (O.R. = 0.16, CI95% = 0.03-0.78, p = 0.023) and LDL-cholesterol (> 130 mg/dL) (O.R. = 0.10, CI95% = 0.02-0.60, p = 0.012) in the control group (data not shown). On the other hand, APOB D allele was associated with less chance of an increase in LDL-cholesterol (> 130 mg/dL) (O.R. = 0.16, CI95% = 0.03-0.94, p = 0.042) in individuals with xanthelasma (data not shown).

Discussion

In this study, the APOE, APOB and LDLR polymorphisms and serum levels of TC, HDL-cholesterol and LDL-cholesterol were similarly distributed in individuals with xanthelasma without co-morbidities and controls. This reinforces the possibility of local lipid-profile alterations with a deposit of lipids only at dermatological sites (Bergman, 1994; Bergman et al., 1996).

In our series, the allelic and genotypic distributions of APOE HhaI polymorphism were similar in individuals with xanthelasma and controls, and showed no significant association to lipid profiles in either group. These results are similar to those reported by Gomez et al. (1988) and Lin et al. (2004). On the other hand, they differ from those by Ribera et al. (1995), who detected a significantly increased frequency of the E3E4 genotype in patients with xanthomatosis together with an altered lipid profile. Moreover, the presence of the APOE*2 allele was shown to be a factor related to significantly higher levels of TG and lower levels of TC and LDL-cholesterol in control individuals compared to patients (Eto et al., 1986). This intensifies controversy regarding the protective effect against cardiovascular manifestations conferred by this allele (Yang et al., 2004).

This study showed similarities between the groups when considering allelic frequencies and genotypic combinations of APOB XbaI and Ins/Del gene polymorphisms. The genotypic distribution observed for APOB XbaI was found to be just marginally different (p = 0.02-0.05) from that expected within the HWE hypothesis in the group of patients.

Multivariate logistic regression analysis with stepwise criteria revealed an association between the presence of the APOB X+ allele and less chance of an increase in TC and LDL-cholesterol in the control group, this being contrary to results as reported by Series et al. (1993).

Brazilian individuals with xanthelasma, and who were carriers of the APOB D allele, presented less chance of undergoing an increase in LDL-cholesterol, this suggesting its association with the lipid profile, as already reported by Hansen et al. (1993) in Denmark. However, no associations were detected through other studies (Gaffney et al., 1993; Jemaa et al. 2004).

LDLR AvaII and HincII polymorphisms did not discriminate groups by their allelic or genotypic distribution, nor did they exert any influence on the lipid profile. Controversial results were reported in Thai subjects by Pongrapeeporn et al. (2000), when considering the presence of the A+A+ genotype associated to higher TC and TG, and lower HDL-cholesterol levels. Additionally, Salazar et al. (1999) demonstrated increased TC and LDL-cholesterol levels in Brazilian carriers of the H+H+ genotype.

In conclusion, our data show that apoE, apoB and LDLR polymorphisms are not associated with a predisposition for xanthelasma. However, distinct APOB polymorphisms are highlighted as risk factors for alterations of the lipid profile in individuals with xanthelasma (APOB D) and controls (APOB X+).

Acknowledgments

This study was supported by grants from FAPESP - Brazil (02/02545-0). The authors would like to thank Helymar da Costa Machado, José Antônio Cordeiro and Moacir Fernandes de Godoy for their assistance with the statistical analysis, and David Andrew Hewitt for reviewing the English text. Marcelo A. Nakazone was the recipient of a fellowship from FAPESP-Brazil (02/02544-3). At present, M.A. Nakazone and A. Pinheiro are recipients of fellowships from FUNDAP-Brazil and R.D.C. Hirata and M.H. Hirata from CNPq-Brazil.

Received: May 16, 2008; Accepted: November 4, 2008.

Associate Editor: Paulo A. Otto

• Allain CC, Poon LS, Chan CSG, Richmond W and Fu PC (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20:470-475.
• Bergman R, Kasif Y, Aviram M, Maor I, Ullman Y, Gdal-On M and Friedman-Birnbaum R (1996) Normolipidemic xanthelasma palpebrarum: Lipid composition, cholesterol metabolism in monocyte-derived macrophages, and plasma lipid peroxidation. Acta Derm Venereol 76:107-110.
• Bergman T (1994) The pathogenesis and clinical significance of xanthelasma palpebrum. J Am Acad Dermatol 30:236-241.
• Boerwinkle E, Lee SS, Butler R, Schumaker VN and Chan L (1990) Rapid typing of apolipoprotein B DNA polymorphisms by DNA amplification. Atherosclerosis 81:225-232.
• Breier F, Zegler B, Reiter H, Gschnait F and Zegler BW (2002) Papular xanthoma; a clinicopathological study of 10 cases. J Cutan Pathol 29:200-206.
• Bucolo G and David H (1973) Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem 19:476-482.
• Cavalli SA, Hirata MH, Salazar LA, Diament J, Forti N, Giannini SD, Nakandakare ER, Bertolami MC and Hirata RD (2000) Apolipoprotein B gene polymorphisms: Prevalence and impact of serum lipid concentrations in hypercholesterolemic individuals from Brazil. Clin Chim Acta 302:189-203.
• Da Luz PL, Cesena FH, Favarato D and Cerqueira ES (2005) Comparison of serum lipid values in patients with coronary artery disease at < 50, 50 to 59, 60 to 69, and > 70 years of age. Am J Cardiol 96:1640-1643.
• Eto M, Watanabe K and Ishii K (1986) Reciprocal effects of apolipoprotein E alleles (epsilon 2 and epsilon 4) on plasma lipid levels in normolipidemic subjects. Clin Genet 29:477-484.
• Friedewald WT, Levy RI and Fredrickson DS (1972) Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifugation. Clin Chem 18:499-502.
• Gaffney D, Freeman DJ, Shepherd J and Packard CJ (1993) The ins/del polymorphism in the signal sequence of apolipoprotein B has no effect on lipid parameters. Clin Chim Acta 218:131-138.
• Gomez JA, Gonzales MJ, Moragas JM, Serrat J, Gónzales-Sastre F and Pérez M (1988) Arch Dermatol 125:1281-1282.
• Guo Y, Guo J, Zheng D, Pan L, Li Q and Ruan G (2002) Relationship between the Nco I, Ava II polymorphism of low density lipoprotein receptor gene and atherosclerotic cerebral infarction. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 19:209-212.
• Hansen PS, Gerdes LU, Klausen IC, Gregersen N and Faergeman O (1993) Polymorphism in the apolipoprotein B-100 gene contributes to normal variation in plasma lipids in 464 Danish men born in 1948. Hum Genet 91:45-50.
• Hirashiki A, Yamada Y, Murase Y, Suzuki Y, Kataoka H, Morimoto Y, Tajika T, Murohara T and Yokota M (2003) Association of gene polymorphisms with coronary artery disease in low- or high-risk subjects defined by conventional risk factors. J Am Coll Cardiol 42:1429-1437.
• Hixson JE and Vernier DT (1990) Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with Hha I. J Lipid Res 31:545-548.
• Hobbs H, Brown M and Goldstein J (1992) Molecular genetics of the LDL receptor gene in familial hypercholesterolemia. Hum Mutat 1:445-466.
• Jemaa R, Mebazaa A and Fumeron F (2004) Polymorphism insertion/deletion of apolipoprotein B gene: Effect on lipid levels in obese patients. Ann Biol Clin 62:183-188.
• Lin SK, Kao JT, Tsai SM, Tsai LY, Lin MN, Lai CJ and Zhong WL (2004) Association of apolipoprotein E genotypes with serum lipid profiles in a healthy population of Taiwan. Ann Clin Lab Sci 34:443-448.
• Norman D, Sun XM, Bourbon M, Knight BL, Naoumova RP and Soltar AK (1999) Characterization of a novel cellular defect in patients with phenotypic homozygous familial hypercholesterolemia. J Clin Invest 104:619-628.
• Pongrapeeporn KU, Pimsawat T, Likidlilid A, Poldee S, Yamwong P, Amomrattana A and Ong-Ajyooth S (2000) Effect of Ava II and Nco I polymorphisms at the low density lipoprotein receptor gene on plasma lipid levels in a group of Thai subjects. J Med Assoc Thai 83:74-80.
• Renges HH, Wile DB, McKeigue PM, Marmot MG and Humphries SE (1991) Apolipoprotein B gene polymorphism are associated with lipid levels in men of south Asian descent. Atherosclerosis 91:267-275.
• Ribera M, Pintó X, Argimon JM, Fiol C, Pujol R and Ferrándiz C (1995) Lipid metabolism and apolipoprotein E phenotypes in patients with xanthelasma. Am J Med 99:485-490.
• Rios DL, Vargas AF, Torres MR, Zago AJ, Callegari-Jacques SM and Hutz MH (2003) Interaction between SREBP-1a and APOB polymorphisms influences total and low-density lipoprotein cholesterol levels in patients with coronary artery disease. Clin Genet 63:380-385.
• Salazar LA, Hirata MH, Giannini SD, Forti N, Diament J, Issa JS and Hirata RD (1999) Effects of Ava II and Hinc II polymorphisms at the LDL receptor gene on serum lipid levels of Brazilian individuals with high risk for coronary heart disease. J Clin Lab Anal 13:251-258.
• Salazar LA, Hirata MH, Giannini SD, Forti N, Diament J, Lima TM and Hirata RD (2000) Seven DNA polymorphisms at the candidate genes of atherosclerosis in Brazilian women with angiographically documented coronary artery disease. Clin Chim Acta 300:139-149.
• Santos RD and Sociedade Brasileira de Cardiologia (2001) III Brazilian Guidelines on Dyslipidemias and Guideline of Atherosclerosis Prevention from Atherosclerosis Department of Sociedade Brasileira de Cardiologia. Arq Bras Cardiol 77:1-48.
• Series JJ, Gaffney D, Packard CJ and Shepherd J (1993) Frequency of the Xba I, EcoR I, Pvu II and MSp I polymorphism of the apolipoprotein B gene in relation to hypercholesterolemia in the general population. Clin Chim Acta 215:89-98.
• Souza DR, Nakachima L, Biagioni RB, Nakazone MA, Pinhel MA, Trindade DM, Mafra VT, Tácito LH, Martin JF, Pinheiro Junior S, et al. (2007) Relevance of apolipoprotein E4 for the lipid profile of Brazilian patients with coronary artery disease. Braz J Med Biol Res 40:189-197.
• Sugiyama N, Marcovina S, Gown AM, Seftel H, Joffe B and Chait A (1992) Immunohistochemical distribution of lipoprotein epitopes in xanthomata from patients with familial hypercholesterolemia. Am J Pathol 141:99-106.
• Tursen U, Eskandari G, Kaya TI, Tamer L, Ikizoglu G and Atik U (2006) Apolipoprotein E polymorphism and lipoprotein compositions in normolipidaemic xanthelasma patients. J Eur Acad Dermatol Venereol 20:260-263.
• Yang SL, He BX, Liu HL, He ZY, Zhang H, Luo JP, Hong XF and Zou YC (2004) Apolipoprotein E gene polymorphisms and risk for coronary artery disease in Chinese Xinjiang Uygur and Han population. Chin Med Sci J 19:150-154.

Publication Dates

• Publication in this collection
  06 Mar 2009
• Date of issue
  2009

History

• Accepted
  04 Nov 2008
• Received
  16 May 2008