J Periodont Res 2015; 50: 509–518 © 2014 John Wiley & Sons A/S. All rights reserved Published by John Wiley & Sons Ltd

JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12237

H. Gao*, Y. Tian*, H. Meng, J. Hou, Associations of L. Xu, L. Zhang, D. Shi, R. Lu, X. Feng, X. Wang, Z. Chen Department of Periodontology, Peking E and University School and Hospital of Stomatology, Beijing, China low-density -related 5 polymorphisms with dyslipidemia and generalized aggressive periodontitis in a Chinese population

Gao H, Tian Y, Meng H, Hou J, Xu L, Zhang L, Shi D, Lu R, Feng X, Wang X, Chen Z. Associations of and low-density lipoprotein receptor- related protein 5 polymorphisms with dyslipidemia and generalized aggressive periodontitis in a Chinese population. J Periodont Res 2015; 50: 509–518. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Background and Objective: Dyslipidemia is associated with aggressive periodonti- tis, a condition characterized by the rapid destruction of the periodontium. Apo- lipoprotein E (APOE) and low-density lipoprotein receptor-related protein 5 (LRP5) are involved in immunomodulation and inflammatory activity. We eval- uated the association of LRP5 and APOE polymorphisms with serum con- centrations and generalized aggressive periodontitis within a Chinese population. Material and Methods: Mean serum lipid concentrations were compared across LRP5 and APOE polymorphisms, among cases (n = 185) and controls (n = 138). Multivariable logistic regression was used to evaluate the independent and combined associations of LRP5 and APOE polymorphisms with generalized Huanxin Meng, BDS, MS, PhD, Peking aggressive periodontitis. University School and Hospital of Stomatology, 22, Zhongguancun Nandajie, Hai Dian District, Results: Compared with controls, individuals with generalized aggressive peri- Beijing 100081, China odontitis exhibited significantly lower serum total (TC) and lower Tel: +86 10 82195522 high-density lipoprotein cholesterol (HDL-c). Individuals with LRP5 polymor- Fax: +86 10 62173402 e-mail: [email protected] phisms (rs682429-AA or rs312016-GG) exhibited higher TC, higher HDL-c and decreased odds for generalized aggressive periodontitis. Haplotype (A-G), deter- *These authors contributed equally to this mined by rs682429 and rs312016, was also associated with decreased odds for study. generalized aggressive periodontitis. Furthermore, individuals with the combined Key words: apolipoprotein E; generalized polymorphisms (LRP5-rs682429-AA and APOE-rs429358-CC/CT) had higher aggressive periodontitis; low-density lipoprotein receptor-related protein 5; serum lipid; single levels of low-density lipoprotein cholesterol, higher levels of TC and decreased nucleotide polymorphisms odds for generalized aggressive periodontitis. Accepted for publication August 23, 2014 510 Gao et al.

Conclusion: Independently or combined with APOE, LRP5 polymorphisms may lead to dyslipidemia and are associated with generalized aggressive periodontitis. Dyslipidemia may be a risk indicator for generalized aggressive periodontitis in the Chinese population. Furthermore, two LRP5 polymorphisms (rs682429 and rs312016) might be useful for identifying subjects at higher risk of generalized aggressive periodontitis.

Aggressive periodontitis is an uncom- APOE binds to cholesterol to form a As far as the researchers are aware, mon and severe form of periodontitis, highly hydrophilic lipoprotein, allow- no published studies have evaluated characterized by the rapid destruction ing efficient transportation in the the independent or combined associa- of the periodontium in otherwise blood (16), and mediates the binding tion of APOE and LRP5 polymor- healthy adolescents and young adults between the lipoprotein and the low- phisms with generalized aggressive (1). Although microbes are often cited density lipoprotein receptor (17). Of periodontitis. The purpose of the cur- as the primary causative agent of all the different APOE isoforms, the rent study is two-fold: (i) to evaluate aggressive periodontitis, the manifes- APOE e4 isoform is that which is the relationship between the APOE tation and progression of the disease most strongly associated with higher and LRP5 polymorphisms and serum results from the complex interactions lipid levels and increased risk of car- lipid concentrations; and (ii) to evalu- between oral bacteria, the host diovascular disease (18). Furthermore, ate the independent and combined response and behavioral factors. It APOE is involved in immunomodula- association of APOE (rs429358, has also been suggested that genetic tion and inflammatory activity (19); rs7412) and LRP5 (rs312016, factors seem to play an important role APOE polymorphisms have been rs682429) polymorphisms with gener- in predisposing individuals to peri- associated with a range of inflamma- alized aggressive periodontitis in a odontitis, which is supported by sev- tory and autoimmune diseases and Chinese population. eral genetic segregation analyses of disorders, including Alzheimer’s dis- families and a twin study of chronic ease (20), diabetic nephropathy (21), Material and methods periodontitis (2,3). severe hepatitis C infection (22), high Evidence is building that hyperlipid- levels of C-reactive protein (23) and Study population emia is a risk indicator for periodontal rheumatoid arthritis (16). disease (4–6). Studies have found that Similarly, low-density lipoprotein A total of 323 unrelated individuals severe periodontitis is associated with receptor-related protein 5 (LRP5) of Chinese ethnicity volunteered to lower levels of high-density lipopro- plays a regulatory role in cholesterol participate in this case–control study. tein cholesterol (HDL-c), higher levels metabolism (24) and is involved in the Each case received a full periodontal of low-density lipoprotein cholesterol clearance of APOE-containing lipo- examination and a set of full-mouth (LDL-c) and significantly higher levels (25). Recent studies have radiographs were obtained. Two of plasma (7–9). How- shown that APOE/LRP5 double- senior, experienced periodontists, who ever, contradictory findings have also knockout mice exhibited higher were calibrated, carried out all clinical been reported (10–12). plasma cholesterol levels that were examinations and diagnosis. The Nutrition is also an important risk 60% higher than the levels among kappa value of intrarater agreement factor for periodontitis, as poor nutri- APOE knockout mice (26,27). Fur- for the diagnosis of generalized tion may inhibit the immune response thermore, LRP5 is also a co-receptor aggressive periodontitis was estimated (13). Specifically, foods high in dietary of Wnt/b- signaling (28), a for 50 individuals, whilst the kappa cholesterol or fatty acids may inhibit mechanism that might be involved in value for inter-rater agreement for the the immune response and accelerate the development of periodontitis (29). diagnosis of generalized aggressive the bactericidal effect on Porphyro- LRP5 polymorphisms have been asso- periodontitis was estimated for 20 monas gingivalis (14). Although the ciated with cholesterol level (30), bone individuals. The kappa values for in- importance of nutrition on the mineral density (31), trarater/inter-rater agreement were immune function has been estab- (32), diabetes mellitus (33), obesity 0.98 and 0.95, respectively. Patients lished, it remains unclear whether (34), bone fracture (35), Alzheimer’s with generalized aggressive periodon- abnormalities in lipid metabolism or disease (36) and tumors (28,37,38). As titis (cases) (n = 185) were recruited conditions related to dyslipidemia a result of the established relationship from the Clinic of Periodontology lead to periodontal disease or if peri- between periodontitis and osteoporo- Department, Peking University odontal disease leads to impaired lipid sis, diabetes mellitus and obesity School and Hospital of Stomatology, metabolism (15). (15,38), we suspect that LRP5 poly- from July 2001 to December 2012. Apolipoprotein E (APOE) plays an morphisms might also be associated We used the 1999 International Clas- important role in the uptake of . with periodontitis. sification of Periodontal Diseases and APOE, LRP5 polymorphisms, generalized aggressive periodontitis and serum lipid 511

Conditions (1) as the diagnostic crite- terol (TC), , HDL-c and MASSARRAY TYPER software version 3.4 ria for generalized aggressive peri- LDL-c levels, were measured using a (Sequenom). As a quality check of odontitis, which includes all of the biochemical analyzer (Hitachi 7060; our data, we sequenced more than following characteristics: (i) under Hitachi, Tokyo, Japan); fasting serum 5% of the samples (eight cases and 35 years of age; (ii) at least six teeth glucose levels were also measured using seven controls) using an ABI 3100 (at least three of which were not first a biochemical analyzer. DNA sequencer (Applied Biosystems, molars or incisors) with a probing Foster City, CA, USA); the genotype depth of ≥ 5 mm and clinical attach- concordance rate between duplicate Genotyping ment loss of ≥ 3 mm; (iii) no peri- samples was 100%. odontal treatments within the past The SEQUENOM MassARRAY 12 mo; (iv) female cases were not matrix-assisted laser desorption ioniza- Statistical analyses pregnant or lactating; and (v) clini- tion time-of-flight (MALDI-TOF) cally healthy, except for the presence mass spectrometry platform was used Preliminary analysis explored differ- of periodontitis. The patients with to genotype the LRP5 (rs682429, ences in participant characteristics generalized aggressive periodontitis rs312016) and APOE (rs429358, between the case group and control had 22.80 4.91 (range, 10–28) teeth rs7412) polymorphisms (Sequenom, using t-tests and ANOVA for contin- that met the inclusion criterion. Eight San Diego, CA, USA). Primers for the uous variables and chi-square tests for cases over 35 years of age were also PCR and single base extension were categorical variables. Multiple linear included because of their diagnosis of designed using the ASSAY DESIGNER regression was used to compare the aggressive periodontitis when younger software package (Sequenom). serum lipid concentration between the than 35 years of age. Multiplex PCR was performed in 5- case group and the control group and Controls (n = 138) were selected lL volumes containing 0.5 units of among subjects with different geno- from individuals undergoing regular HotStar Taq polymerase (Qiagen, types after adjusting for major con- physical check-ups. To be considered Hilden, Germany), 10 ng of whole- founders (age, sex, BMI and smoking a control, the individual must not genome-amplified genomic DNA, status). In order to establish genetic have had any previous or existing 5 pmol of each primer and 5 lmol of inference of our data, Hardy–Wein- clinical evidence of periodontitis (e.g. deoxynucleotide triphosphates berg equilibrium (HWE) testing was probing depth ≤ 3 mm; clinical (dNTPs). Thermocycling was carried carried out for the LRP5 (rs312016, attachment distances ≤ 1 mm from out at 94°C for 15 min followed by 45 rs682429) and APOE (rs429358, the cemento–enamel junction; and cycles each at 94°C for 20 s, 56°C for rs7412) polymorphisms. < 10% of sites with a bleeding index 30 s and 72°C for 1 min, followed by Multivariate logistic regression of ≥ 2) or a familial history of severe a final incubation at 72°C for 3 min. analysis was performed to test the periodontitis or a known systemic dis- Unincorporated dNTPs were deac- association between LRP5 polymor- order (e.g. atherosclerosis, diabetes, tivated using 0.3 units of shrimp alka- phisms, APOE polymorphisms and rheumatoid arthritis) that could affect line phosphatase (Sequenom) followed aggressive periodontitis, after adjust- the periodontal conditions. Smoking by primer extension using 5.6 pmol of ing for major covariates (age, sex, status was assessed by questionnaires. each primer-extension probe, 50 lmol BMI and smoking status). Pairwise Height and body weight were mea- of the appropriate dNTP/dideoxynu- linkage disequilibrium values (D0 and 2 sured and body mass index (BMI) cleotide triphosphate (ddNTP) combi- r ) were calculated using HAPLOVIEW was calculated accordingly. nation, and 0.5 units of iPLEX version 4.1 (Broad Institute, Cam- (Sequenom). The extension bridge, MA, USA) for markers in reactions were carried out in the fol- LRP5. The statistical power was cal- Isolation of genomic DNA and blood lowing order: one cycle at 94°C for culated using PASS version 11.0 examination 30 s; one cycle at 94°C for 5 s; five (NCSS, LLC., Kaysville, UT, USA). A peripheral blood sample, divided cycles at 52°C for 5 s; and five cycles Prior to statistical analysis, variables into two tubes, was obtained through at 80°C for 5 s; followed by a final of a skewed distribution were logarith- venipuncture between 8:00 h and incubation at 72°C for 3 min. A cat- mically transformed to meet the 10:00 h, following a fasting period. The ion exchange resin was used to assumption of normality; estimates tube containing EDTA was used for remove residual salt from the reac- were subsequently back-transformed genomic DNA isolation; the other tube tions. Purified primer-extension reac- for presentation in the tables. A type I was used for measurement of serum tion products were spotted onto a error level was set a priori at 0.05. Pre- lipid concentrations and fasting serum 384-well spectroCHIP by a MassAR- liminary results were presented in glucose. Genomic DNA was extracted RAY Nanodispenser and determined terms of mean SD or interquartile from each sample using a blood DNA by MALDI-TOF mass spectrometry. ranges for variables that did not meet mini kit (Watson Biotechnologies, Inc., Genotype calling was performed in the assumption of normality. Categori- Shanghai, China), following the manu- real time with MASSARRAY RT software cal variables were presented as counts facturer’s instructions. Serum lipid version 3.0.0.4 (Sequenom, San and percentages. Results were pre- concentrations, including total choles- Diego, CA, USA) and analyzed using sented in terms of adjusted odds ratios 512 Gao et al.

Table 1. Demographic characteristics of the aggressive periodontitis cases and controls 28.65 mg/dL, p = 0.021; HDL-c: 57.82 12.15 mg/dL vs. 63.04 Cases (patients with = generalizedaggressive 15.45 mg/dL, p 0.015; fasting blood periodontitis) Controls glucose: 94.01 9.54 mg/dL vs. Variable (n = 185)a (n = 138)a p-value 88.48 9.14 mg/dL, p < 0.001). Cases with generalized aggressive periodonti- Age (years) tis tended to have a higher serum tri- Mean SD 27.4 5.1 28.6 7.2 0.095b – – glyceride level compared with controls Range 14 48 20 50 Sex (80.70 1.63 mg/dL vs. 72.33 1.57 Male 75 (40.5) 56 (40.6) mg/dL, p = 0.059). Female 110 (59.5) 82 (59.4) 0.994c For all the single-nucleotide poly- Smoking status morphisms (SNPs), genotype call Nonsmokers 164 (88.6) 135 (97.8) rates were > 98% and minor allele c Current smokers 21 (11.4) 3 (2.2) 0.002 frequencies were > 0.01. No evidence BMI of deviation from Hardy–Weinberg 18.5–24.9 127 (68.6) 119 (86.2) < 18.5 23 (12.4) 11 (14.5) equilibrium was found for all investi- ≥ 25.0 35 (18.9) 8 (5.8) 0.001c gated SNPs (p > 0.05). Two SNPs in LRP5 were in strong linkage disequi- BMI, body mass index. librium (D0 = 0.99, r2 = 0.96). Our aData are presented in terms of mean SD or total n (%). bComparison of age between cases (patients with generalized aggressive periodontitis) and sample size provided the power of controls, performed using a two sample t-test. 0.86, 0.86, 0.80 and 0.74 to detect an cComparison of sex, smoking status and BMI between cases (patients with generalized effective size of odds ratio = 0.4 for aggressive periodontitis) and controls, performed using a chi-square test. the rs682429, rs312016, rs429358 and rs7412, respectively. (AORs) and 95% confidence intervals generalized aggressive periodontitis There were significant differences in (CIs). Analyses were performed using and controls are displayed in Table 2. the distributions of LRP5 and APOE SPSS version 17.0 (IBM Co., Armonk, The mean values of the periodontal polymorphisms between cases with NY, USA). parameters were higher among the generalized aggressive periodontitis cases than the controls. and controls, after adjusting for age, Compared with controls, cases sex, BMI and smoking status in the Ethical considerations exhibited significantly lower TC, multivariate logistic regression model This study received ethical approval reduced HDL-c and higher fasting (Table 3). Individuals with the LRP5- from the Ethics Committee of Peking serum glucose levels after adjusting for rs312016-GG genotype had decreased University Health Science Center. age, sex, BMI and smoking status (TC: odds (AOR = 0.44, 95% CI: 0.23– Written informed consent was obtained 163.38 32.91 mg/dL vs. 171.95 0.83) for generalized aggressive from all the participants, in accordance with the Declaration of Helsinki. Table 2. Mean serum lipid concentrations and periodontal parameters according to gener- alized aggressive periodontitis status

Results Cases (patients withgeneralized Demographic characteristics of the aggressive cases with generalized aggressive peri- Characteristics periodontitisN1 ControlsN2 p-value p-valuea odontitis and the controls are pre- TC (mg/dL) 163.38 32.91, 179 171.95 28.65, 96 0.032b 0.021 sented in Table 1. No significant LDL-c (mg/dL) 90.61 28.76, 149 95.46 22.34, 78 0.163b 0.306 difference of age and sex was detected HDL-c (mg/dL) 57.82 12.15, 149 63.04 15.45, 78 0.011b 0.015 by an independent t-test and the chi- TG (mg/dL) 80.70 1.63, 180 72.33 1.57, 96 0.069b 0.059 square test. The proportion of smok- Glucose (mg/dL) 94.01 9.54, 181 88.48 9.14, 98 < 0.001b < 0.001 c ing adults was significantly higher Mean PD (mm) 4.88 1.00, 181 1.84 0.41, 135 < 0.001 NA < c among the cases than the controls Mean BI 3.51 0.46, 169 1.09 0.34, 134 0.001 NA (proportions 11.4% vs. 2.2%, respec- Data are presented as Mean SD, n. tively; p = 0.002). The proportion of BI, bleeding index; BMI, body mass index; HDL-c, high-density lipoprotein cholesterol, individuals with a BMI of ≥ 25.0 was LDL-c, low-density lipoprotein cholesterol; N1, number of cases; N2, number of controls; significantly higher among the cases NA, not accessible; PD, probing depth; TC, total cholesterol; TG, triglycerides. aAdjusted for sex (male, female), age (tertile), smoking status (no, yes) and BMI (BMI than the controls (proportions 18.9% < ≥ = 24.9, BMI 25.0). vs. 5.8%, respectively; p 0.001). bComparison of mean serum lipid concentrations, metabolic markers between generalized The mean serum lipid concentra- aggressive periodontitis cases and controls performed using an independent t-test. tions, fasting serum glucose and peri- cComparison of mean bleeding index and probing depth between generalized aggressive odontal parameters among cases with periodontitis cases and controls performed using a Mann–Whitney U-test. APOE, LRP5 polymorphisms, generalized aggressive periodontitis and serum lipid 513

Table 3. Associations of low-density lipoprotein receptor-related protein 5 (LRP5) poly- adjusting for age, sex, BMI and morphisms with generalized aggressive periodontitis smoking status (AOR = 0.15, 95% – = Cases (patients CI: 0.03 0.92; AOR 0.45, 95% CI: – with generalized 0.22 0.92, respectively). aggressive Figure 1 illustrates the differences SNP periodontitis)a Controlsa Crude OR (95% CI) AORb (95% CI) in mean serum lipid concentrations between individuals with different rs312016 LRP5 genotypes (LRP5-rs682429-AA AA/AG 159 (85.9) 103 (75.2) 1 [reference] 1 [reference] GG 26 (14.1) 34 (24.8) 0.50 (0.28–0.87)* 0.44 (0.23–0.83)* vs. GG/AG; LRP5-rs312016- GG vs. rs682429 AA/AG), among cases with general- GG/AG 160 (86.5) 104 (75.4) 1 [reference] 1 [reference] ized aggressive periodontitis and con- AA 25 (13.5) 34 (24.6) 0.48 (0.27–0.45)* 0.42 (0.22–0.81)* trols. In general, individuals with rs429358 LRP5-rs682429-AA or LRP5- TT 159 (87.8) 112 (81.2) 1 [reference] 1 [reference] rs312016-GG exhibited higher TC CC/CT 22 (12.2) 26 (18.8) 0.60 (0.32–1.11) 0.49 (0.24–0.99)* and HDL-c levels than did those with rs7412 CC 155 (84.7) 112 (83.0) 1 [reference] 1 [reference] LRP5-rs682429-GG/AG or LRP5- TT/CT 28 (15.3) 23 (17.0) 0.88 (0.48–1.61) 0.97 (0.50–1.88) rs312016-AA/AG polymorphisms. Figure 2 illustrates the differences AOR, adjusted odds ratio; OR, odds ratio; SNP, single-nucleotide polymorphism. in mean serum lipid concentrations *Significant at p = 0.05. aValues represent n (%). between individuals with different bAdjusted for sex (male, female), age (tertile), smoking status (no, yes) and body mass combined genotypes among cases with index (BMI) (BMI < 24.9, BMI ≥ 25.0). generalized aggressive periodontitis and controls. Individuals with the periodontitis compared with individu- Table 4 demonstrates that individu- combined genotype of LRP5- als with the LRP5-rs312016-AA/AG als with the LRP5 haplotype rs682429-AA and APOE-rs429358- genotype, after adjusting for age, sex, (rs682429-rs312016: A–G) had CC/CT exhibited significantly higher BMI and smoking status. Individuals decreased odds for generalized aggres- TC and LDL-c levels compared with with the LRP5-rs682429-AA polymor- sive periodontitis compared with indi- individuals with the LRP5-rs682429- phism had decreased odds viduals with the LRP5 haplotype GG/AG genotype and the APOE- (AOR = 0.42, 95% CI: 0.22–0.81) for (rs682429-rs312016: G–A) after rs429358-TT genotype. generalized aggressive periodontitis adjusting for age, sex and smoking compared with individuals with the status (AOR = 0.59, 95% CI: 0.41– LRP5-rs682429-GG/AG polymor- 0.84). Discussion phism, after adjusting for age, sex, Individuals with the combined Four important findings emerged BMI and smoking status. Individuals genotype (LRP5-rs682429-AA + from our study: (i) compared with with APOE-rs429358-CC/CT had APOE-rs429358-CC/CT and LRP5- controls, individuals with generalized decreased odds (AOR = 0.49, 95% rs682429-AA + APOE-r429358-TT) aggressive periodontitis exhibited sig- CI: 0.24–0.99) for generalized aggres- had decreased odds of generalized nificantly lower serum TC and lower sive periodontitis compared with indi- aggressive periodontitis compared HDL-c; (ii) individuals with LRP5 viduals with the APOE-rs429358-TT with individuals with the LRP5- SNPs (rs682429-AA or rs312016-GG) polymorphism, after adjusting for rs682429-GG/AG genotype and the exhibited higher TC, higher HDL-c age, sex, BMI and smoking status. APOE-rs429358-TT genotype, after and decreased odds for generalized aggressive periodontitis; (iii) individu- Table 4. Distribution of low-density lipoprotein receptor-related protein 5 (LRP5) haplo- als with combined polymorphisms type (rs682429-rs312016) frequencies and odds ratios (ORs) for generalized aggressive peri- (LRP5-rs682429-AA and APOE- odontitis rs429358-CC/CT) had higher serum Cases (patients LDL-c and TC levels, as well as with generalized decreased odds for generalized aggressive aggressive periodontitis; and (iv) indi- a a b Haplotype periodontitis) Controls Crude OR (95% CI) AOR (95% CI) viduals with the LRP5 haplotype – Total 365 273 (rs682429-rs312016: A G) had G–A 226 (61.9) 140 (51.3) 1 [reference] 1 [reference] decreased odds for generalized A–G 139 (38.1) 133 (48.7) 0.65 (0.47–0.89)* 0.59 (0.41–0.84)* aggressive periodontitis. Our findings build on previous lipid research and AOR, adjusted odds ratio. * contribute to the limited body of Significant at p = 0.05. aValues represent n (%). knowledge of serum lipid concentra- bAdjusted for sex (male, female), age (tertile), smoking status (no, yes) and body mass tions and the LRP5 and APOE index (BMI) (BMI < 24.9, BMI ≥ 25.0). polymorphisms. 514 Gao et al.

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Fig. 1. Comparison of serum lipid concentrations between individuals with different genotypes (rs682429-AA vs. GG/AG; rs312016-GG vs. AA/AG), among cases (patients with generalized aggressive periodontitis) and controls. Serum lipid concentrations of total cholesterol (TC) (A), triglycerides (TG) (B), low-density lipoprotein cholesterol (LDL-c) (C) and high-density lipoprotein cholesterol (HDL-c) (D) were compared between individuals with different genotypes (rs682429-AA vs. GG/AG; rs312016-GG vs. AA/AG, respectively) among generalized aggressive periodontitis cases and controls after adjusting for sex (male, female), age (tertile), smoking status (no, yes) and body mass index (BMI) (BMI < 24.9, BMI ≥ 25.0). Results are displayed in terms of means SD; statistical significance was determined using multiple linear regression (*p < 0.05). GAP, generalized aggressive periodontitis.

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Fig. 2. Comparison of serum lipid concentrations between individuals with different combined genotypes (rs682429-AA+rs429358-CC/CT vs. rs682429-AA+rs429358- TT vs. rs682429-GG/AG+rs429358-CC/CT vs. rs682429-GG/AG+rs429358-TT), among cases (patients with generalized aggressive periodontitis) and controls. Serum lipid concentrations of total cholesterol (TC) (A), triglycerides (TG) (B), low- density lipoprotein cholesterol (LDL-c) (C) and high-density lipoprotein cholesterol (HDL-c) (D) were compared between individuals with different combined genotypes (rs682429-AA+rs429358-CC/CT vs. rs682429-AA+rs429358-TT vs. rs682429-GG/AG+rs429358 -CC/CT vs. rs682429- GG/AG+rs429358-TT) among generalized aggressive periodontitis cases and controls after adjusting for sex (male, female), age (tertile), smoking status (no, yes) and body mass index (BMI) (BMI < 24.9, BMI ≥ 25.0). Results are displayed in terms of means SD; statistical significance was determined using multiple linear regression (*p < 0.05, **p < 0.01). APOE, LRP5 polymorphisms, generalized aggressive periodontitis and serum lipid 515

Inflammation and infection are two periodontitis, as preliminary findings APOE polymorphisms with serum processes that frequently induce dysli- from our study population found sys- lipid concentrations across various pidemia (39). Biomarkers of inflam- temic inflammation (49–51) among study populations (55,56), including mation, including tumor necrosis the aggressive periodontitis cases. Chinese populations (57–59). Earlier factor, interleukin-2 and interferon- Additionally, differences in the age studies showed that APOE e2 reduced gamma, have been shown to increase and race/ethnicity distributions of the levels of TC and LDL-c, whilst serum triglyceride levels and decrease these samples could have also contrib- APOE e4 increased the levels of TC serum cholesterol (40). Additionally, uted to these discrepancies. and LDL-c (60). Recently, a meta- lipopolysaccharide injection (41) and In our study, we observed a signifi- analysis revealed that there was severe sepsis (42) reduce LDL-c and cant association between two LRP5 approximately linear relationships of HDL-c levels. Dyslipidemia is often SNPs (rs682429, rs312016) or LRP5 APOE polymorphisms (when ordered observed among individuals with haplotypes and generalized aggressive e2/e2, e2/e3, e2/e4, e3/e3, e3/e4, e4/e4) chronic inflammatory conditions, such periodontitis, after adjusting for age, with levels of LDL-c and TC and as rheumatoid arthritis and systemic sex and smoking status. Considering with coronary risk (18). Our study lupus erythematosus (43). the functional interaction between builds on previous research by con- Despite this growing understanding APOE and LRP5 genotypes, we firming the combined association of of chronic inflammatory conditions attempted to elucidate the relationship LRP5-rs682429 and APOE-rs429358 and dyslipidemia, the relationship between combined polymorphisms polymorphisms with TC and LDL-c between periodontitis and dyslipide- and generalized aggressive periodonti- levels among a Chinese population of mia is still unclear. Several epidemio- tis. We observed a significant com- generalized aggressive periodontitis logical studies have shown the bined association between APOE and cases and controls. A recent meta- association between hyperlipidemia LRP5 polymorphisms and generalized analysis found that periodontal treat- and periodontal disease. Hyperlipid- aggressive periodontitis, after adjust- ment improved lipid metabolism, emia is a risk indicator for periodon- ing for age, sex, smoking status and especially in those individuals suffer- tal disease (5); specifically, two studies BMI; this finding suggests that the ing from both periodontitis and com- have found higher TC and LDL-c lev- APOE and LRP5 polymorphisms orbidities, such as cardiovascular els among individuals with periodonti- may play a critical role in generalized disease and diabetes mellitus (45). tis (4,6). However, epidemiological aggressive periodontitis. APOE has Therefore, periodontitis might precede evidence has also presented contradic- been shown to play an important role the occurrence of dyslipidemia. tory findings (10,12). In addition, in immune responses (19,52) and in periodontal treatment plays a benefi- the presentation of lipid antigens to Strengths and limitations cial role in lipid metabolism (44,45). immune cells (53), whilst LRP5 is a Our findings showed that patients co-receptor of Wnt/b-catenin signal- The strengths of our study include: with generalized aggressive periodon- ing that has been reported to mediate the novelty of our research question, titis (the cases) exhibited significantly leukocyte-inflammatory responses the consistency in genotyping with lower TC and HDL-c levels than con- (54). previous studies, and the comparabil- trols in a Chinese population. Few Additionally, we observed higher ity of our results. In general, the poly- studies have evaluated the relationship levels of TC and HDL-c levels among morphisms and allele frequencies of between dyslipidemia and generalized individuals with the LRP5-rs682429 both APOE and LRP5 were similar aggressive periodontitis. Rufail et al. AA or LRP5-rs312016 GG polymor- to those of previous studies among (46) observed higher TC and triglycer- phisms. A significant association the Chinese population (61–63). The ide levels among individuals with between the LRP5-rs3736228 (30) and allele APOE e2 has a cytosine-to-thy- aggressive periodontitis compared LRP5-A1330V (31) polymorphisms mine base-pair substitution in rs7412, with controls, whilst Davis et al. (47) and hypercholesterolemia has previ- whilst in APOE e4, a thymine is reported that levels of inflammatory ously been observed. Although the replaced with a cytosine in rs429358 mediators and serum lipid concentra- loci we selected were different from (64). The polymorphisms and allele tions were not significantly different those in the above two studies, our frequencies of both APOE and LRP5 among individuals with aggressive results were consistent with their find- vary considerably across race/ethnic- periodontitis compared with controls. ings, further suggesting that the LRP5 ity. The APOE allele frequencies were Our findings are somewhat compara- polymorphism is significantly associ- similar to that of Japanese (65) and ble to results found in a case–control ated with cholesterol levels. The Korean (65) individuals, but were study of chronic periodontitis among mechanism of action linking LRP5 obviously different to those in subjects a Chinese Han population (48). The and generalized aggressive periodonti- from other countries, such as Mongo- discrepancy between our results and tis probably involves a decrease in lia (66), Malay Aborigines (65), cen- those of other studies might be a hepatic clearance of rem- tral Africa (65), Brazil (67) and result of the poor periodontal condi- nants (26). Northern European countries (65). tion and the severe inflammation in Indeed, there is an abundance of The e3 allele frequency increased in our cases with generalized aggressive research supporting the association of Chinese individuals, whereas the e4 516 Gao et al. allele frequency decreased. In addi- cerides, total cholesterol and fractions. Acknowledgements tion, the two SNPs within LRP5 were Braz Oral Res 2005;19:284–289. in strong linkage disequilibrium, We acknowledge that this work was 11. Korhonen S, Saxlin T, Suominen L, Jula A, Knuuttila M, Ylostalo P. Serum cho- which was consistent with a previous financially supported by the following lesterol ratios and periodontal infection: study (68). The A allele frequency at organizations: the National Natural results of the Health 2000 Survey. J Clin rs682429 in the present study was Science Foundations of China, Beij- Periodontol 2011;38:787–794. lower than that of US Caucasians ing, China (30471882, 30973319, 12. Valentaviciene G, Paipaliene P, Nedzel- (0.481 vs. 0.714), whilst the G allele 81271149); the National Key Project skiene I, Zilinskas J, Anuseviciene OV. frequency at rs312016 was lower than of Scientific and Technical Supporting The relationship between blood serum that of US Caucasians and Italians Programs of China, Beijing, China lipids and periodontal condition. Stom- atologija 2006;8:96–100. (0.485 vs. 0.714 and 0.675, respec- (2007BAI18B02); and the Key Pro- 13. Marti A, Marcos A, Martinez JA. Obes- tively) (34,37,69). gram of Clinical Specialty, National ity and immune function relationships. A limitation exists in the relatively Ministry of Health P.R.C., Beijing, Obes Rev 2001;2:131–140. small sample size after stratifying China. 14. Cutler CW, Iacopino AM. Periodontal according to polymorphisms. disease: links with serum lipid/triglycer- Although we observed statistically sig- ide levels? Review and new data. J Int Acad Periodontol 2003;5:47–51. nificant differences in the TC and References 15. Saito T, Shimazaki Y. Metabolic disor- LDL-c levels across the combined 1. Armitage GC. Development of a classifi- ders related to obesity and periodontal genotype groups, it warrants future cation system for periodontal diseases disease. Periodontol 2000 2007;43:254– investigation with a larger sample size and conditions. Ann Periodontol 266. to verify the results of the present 1999;4:1–6. 16. Maehlen MT, Provan SA, de Rooy DPC study. In addition, the association of 2. 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