Original article
Paraoxonase 1 (PON1) gene -108C>T and p.Q192R polymorphisms and arylesterase activity of the enzyme in patients with dementia

Introduction

Alzheimer disease (AD) is the main type of dementia [1]. In AD, senile plaques with accumulated beta-amyloid and neurofibrils with hyperphosphorylated tau protein as the main component are found [11]. Ano­ther type is dementia of vascular origin (VaD). There is however no sharp limit between these types and many cases are classified as mixed dementia (MD).

Paraoxonase 1 (PON1) is an enzyme of a very broad specificity. Its action is directed against organophosphates (the name paraoxonase originates from this activity), arylesters and lactones. Its important function is to protect lipoproteins, particularly low density lipoproteins (LDL), from oxidative modification. PON1 is transported in serum in association with high density lipoproteins (HDL) [24].

PON1 activity is low in atherosclerosis [23,24]. It was also shown to be low in dementia [25,32].

Human PON1 gene has almost 200 single nucleotide polymorphisms (SNPs). The most important are the following SNPs: -108C>T (rs705379) and -162A>G (rs705381) in the promoter [22] and p.Q192R (rs662) and p.L55M (rs854560) in the coding region of the gene [24]. Para­oxon hydrolytic activity is higher in the case of 192R allele and 55L allele as compared with Q and M ones, respectively [24]. Protection against LDL oxidation is more effective in the case of the 192Q allele [2]. Seve­ral authors reported that when PON1 activity was measured using arylester as a substrate, no differences de­pending on Q192R polymorphism were observed. It was often admitted that paraoxon was a discriminating substrate and phenylacetate was a non-discriminating one [4,9,20,23].

A genetic variability in the promoter has a signifi­cant effect on PON1 level and activity of the C allele be­ing the more active one [3,22].

Multiple studies investigated the influence of particular PON1 alleles on the development of various diseases particularly on cardiovascular disease, however, they did not give definite results [23,31,32].

The results concerning the significance of PON1 gene polymorphism in dementia were also conflicting [5,7, 15,19,26,29,30].

An interesting observation concerns the existing re­lationship between the Q192R polymorphism and res­ponsiveness to cholinesterase inhibitors which is the most established treatment strategy in AD. The carriers of R allele were found to be better responders [27]. This influence of the polymorphism could result from the interaction between PON1 and cholinesterase [4,21].

In our study we determined PON1 activity using phe­nylacetate as a substrate (i.e. its arylesterase activi­ty) and identified both PON1 -108C>T and Q192R polymorphic forms in subjects with dementia and in controls. We also investigated the effect of both polymor­phisms on serum PON1 activity.

Material and methods

The investigated group consisted of 304 individuals with dementia – 108 men and 196 women, mean age 73.3 ± 7.90 years, and a control group of 129 persons – 58 men and 71 women, mean age 71.6 ± 7.26 without symptoms of dementia and in good general health.

Mini Mental State Examination and a clock drawing test were used as screening tests for existing de­mentia. Dementia was diagnosed using ICD-10 and DSM-IV. Patients and controls were subjected to a ge­neral medical and neurological evaluation, CT or MR examinations and neuropsychological tests. The type of dementia was recognized according to NINCDS-ADRDA criteria for Alzheimer’s disease (AD) and NINDS-AIREN criteria for vascular dementia (VaD). Significant radiological evidence of cerebrovascular disease in CT or MRI suggested a coexisting cerebrovascular disease in AD and those patients were included into the mixed dementia (MD) group. 136 individuals were recognized as AD (38 men and 98 women, 72.3 ± 8.45 years), 64 as VaD (29 men and 35 women, 71.8 ± 7.46 years) and 104 as MD (41 men and 63 women, 75.5 ± 6.93 years).

Paraoxonase1 activity was determined spectro­pho­tometrically based on the Kitchen et al. method [18] using phenyloacetate as a substrate. One unit of activity was 1 mol of phenol liberated per minute by 1 ml of serum.

PON1 gene polymorphisms were identified in ge­nomic DNA isolated from blood leukocytes using phe­nol extraction. PON1 gene promoter -108 C>T polymorphism was identified by the Brophy et al., PCR-RFLP method [3] and PON1 p.Q192R polymorphism was investigated by the PCR-RLFP method of Humbert et al. [16] with minor modifications described by Hasselwander et al. [13].

High density lipoprotein cholesterol (HDL-C) was determined after removing apoB containing lipoproteins by precipitation. Cholesterol was determined using the enzymatic method.

Linkage disequilibrium (LD) statistics (D’ and r2) for genetic polymorphisms and the haplotype frequency were determined using the SNPAnalyzer v2.0 (ISTECH Inc, Goyang, Korea) software [34]. All other statistical analyses were performed using Statistica version 9.

The 2 test was used to evaluate the concordance of genotype frequencies with Hardy-Weinberg’s equili­brium expectations. Differences in means between groups were tested using Student t-test or the variance analysis (ANOVA) followed by Dunnet or Bonfferoni post-hoc tests for multiple comparisons. The means were adjusted for age, sex and HDL-C (and PON1-108C>T polymorphism) using the covariance analysis (ANCOVA) with above-mentioned variables as covariates. Statistical significance of the differences in the frequencies of qualitative variables was evaluated using Pearson’s 2 test. The associations between various types of de­mentia and different variables were identified using multiple logistic regression analysis and presented as odds ratios (OR) with 95% confidence intervals (CI). Multivariate stepwise regression analysis was performed to assess the influence of some genetic and non-gene­tic variables on PON1 activity. The following factors were considered as independent variables: age, sex, HDL-C, presence of PON1 -108T allele and presence of PON1 192R allele. P-values lower than 0.05 were considered as statistically significant.

The study was approved by the Ethics Committee of the Institute of Psychiatry and Neurology. Subjects gave their informed consent either directly or through his or her guardian.

Results

PON1 arylesterase activity in dementia and in the control group



The observation that PON1 activity in demented pa­tients, particularly in dementia of a neurodegenerative character, was significantly lower as compared with

controls was made before [32] and confirmed in the present study (Table I). In the present study we found that low PON1 activity i.e. below 135 mU/mL (the lowest quartile of control group) adjusted for age, sex and HDL-C significantly increased risk of dementia (OR = 2.12 [95% CI: 1.29-3.48; p = 0.003]), of AD (OR = 2.11

[95% CI: 1.18-3.76; p = 0.012]) and of MD (OR = 2.54

[95% CI: 1.36-4.75; p = 0.004]) (data not shown).



PON1 polymorphism in dementia and in the control group



The frequencies of PON1 -108C>T and PON1 p.Q192R genotypes and alleles are presented in Table I. In all in­vestigated groups the distributions of genotypes were in Hardy-Weinberg equilibrium.

In the whole dementia group, the prevalence of the T allele was slightly higher than in controls (73.3% vs. 64.4%, p = 0.062). In the AD group, the prevalence

of PON1-108T allele carriers was significantly higher than in controls (75.7% vs. 64.4%, p = 0.043). The frequencies of the variants did not differ significantly between

other investigated groups. These observations were con­firmed by logistic regression analysis. It can be summarized that the dementia risk was 1.52 (OR) [95% CI: 0.98-2.37; p = 0.064] and AD risk 1.73 (OR) [95% CI:

1.01-2.75; p = 0.045] times greater in carriers of the PON1 -108T allele as compared with non-carriers of this allele. The risk was still greater after adjustment of the re­sults for age, sex and HDL-C, i.e. OR = 1.73 [95% CI: 1.09-2.75; p = 0.020] in the whole dementia group

and 2.13 [95% CI: 1.20-3.81; p = 0.018] in AD (data not shown).

The frequencies of the p.Q192R genotypes and alleles did not differ significantly between the investigated groups.

No significant linkage disequilibrium (LD) was observed among the studied PON1 coding and promoter polymorphisms (D’= 0.096 and r2 = 0.0029; p = 0.117).

The results of the haplotype analysis for combination of both PON1 polymorphisms in various groups of demented patients and controls are shown in Fig. 1. The frequency of rare T-R haplotype was significantly higher in the whole dementia group and AD and MD groups as compared with controls. This haplotype was associated with the whole dementia group, as well as AD and MD with odds ratios of 1.72, 1.87 and 1.93, res­pectively.



PON1 polymorphism and activity



The relationship of PON1 activity with PON1 gene polymorphisms in dementia and in the control group is shown in Table II. Both in the whole dementia group and in controls, serum PON1 activity was significantly (p < 0.00001) associated with PON1 -108C>T promoter polymorphism. The highest activities were found in CC and the lowest in TT homozygotes. The statistically significant differences were observed also after adjustment of the means for factors known to affect PON1 activity, i.e. age, sex and HDL-C. A weaker association of PON1 activity concerned PON1 p.Q192R polymorphism. There was a tendency to higher PON1 activity in QQ in comparison with QR and RR genotypes both in the whole dementia group (p = 0.064) and in the control group (p = 0.095). The statistically significant dif­ferences were observed after adjustment of the means for age, sex, HDL-C and presence of PON-108T allele, both in dementia and controls (p = 0.0005 and p = 0.0003, respectively).

The simultaneous effect of both PON1 polymorphisms on enzyme activity in dementia and in controls is illustrated in Fig. 2. In both groups, the -108CC/192QQ ge­notype was associated with the highest, whereas the -108TT/192RR genotype in dementia and 108TT/192QR genotype in controls (-108TT/192RR genotype was ab­sent in the control group) with the lowest arylesterase activity. A comparison of subjects – carriers of these two genotypes revealed an almost two-fold difference in mean arylesterase activity (185 vs. 102 U/mL in the de­mentia group and 208 vs. 111 in controls).

The significant association of PON1 activity with PON1 -108C>T and p.Q192R polymorphisms was confirm­ed in multivariate regression analysis (Table III), in which other factors known to affect PON1 activity, i.e. age, sex and HDL-C were also taken into account. It was shown that the PON1 -108 T allele explained about 40% of the variance of the PON1 activity, both in the whole dementia and control groups (38.6% and 38.0%, respectively). The influence of PON1 192R allele on enzyme activity was considerably lower (R2 = 2.6% in dementia and 6.1% in controls) and was comparable with influence of age and HDL cholesterol (2.6% to 10.8%).

Discussion

The present study confirms our earlier results (performed on smaller groups) that PON1 activity is significantly lower in demented patients, particularly in dementia of a neurodegenerative type as compar­ed with controls [32]. A significant difference was also found in vascular dementia after adjustment of the means for age, sex, and HDL-C. In addition, multiple linear regression analysis demonstrates low arylesterase activity as an independent predictive risk factor for the whole group with dementia, AD and MD.

The comparison of PON1 gene alleles frequency in subjects with dementia and in controls was studied by several authors and showed various results. Dantoine [7] found that the frequency of 192Q allele was lower in dementia of vascular origin (VaD) as compared with the general population but not in Alzheimer’s disease (AD). Pola et al. [26] and also Shi et al. [30] in the Chinese population did not find differences between AD patients and controls. Scacchi et al. [29] as well as He et al. [14] suggested that the RR genotype being less frequent in AD could have a protective role for AD development. Helbecque et al. [15] and Cellini et al.

[5] underlined the importance of promoter -108C>T

poly­­morphism as the less active T allele could cause a great­er risk for AD. In the study of a Polish population group supplemented with a meta-analysis including 1266 patients and 1286 controls, the association of three polymorphisms: the -161C/T in the promoter, the Q192R and the L55M ones with the AD risk was not confirmed [19].

In our results, the -108 polymorphism showed a statistically significant difference in the distribution as compared with controls in the AD group (T allele was more frequent), whereas the Q192R polymorphism did not show any differences between the investigated groups.

We have found an association of the rare T-R haplotype with dementia, particularly with Alzheimer’s disease and mixed dementia. The same haplotype including both -108T and 192R alleles was revealed to be an ‘at-risk haplotype’ by Helbecque et al. in demented non-AD patients [15].

Brophy et al. [3] have shown the existence of considerable linkage disequilibrium between the -108C>T polymorphism and the p.Q192R and p.L55M ones. This fact was confirmed neither by other authors [6,12] nor in our study.

Based on our results, the main conclusion is that PON1 activity has a prevailing influence on the de­mentia risk. A similar view was expressed by Jarvik et al. [17] in the case of factors predicting vascular disease. As it was shown that PON1 gene promoter polymorphism plays a considerable role in PON1 activity, it is obvious that its influence in dementia development particularly in Alzheimer’s disease is also apparent. The p.Q192 polymorphism could play an additional role.

The exact mechanism of paraoxonase1 influence on dementia and AD risk is not fully explained. It was shown that low enzyme activity was associated with the increased oxidative stress, increased risk of cardiovascular disease, stroke and type 2 diabetes [2,17, 23], known dementia risk factors [8]. Recently in auto­psy-confirmed AD study of Leduc et al. [21], PON1 p. L55M and p.Q192R genetic variants were significantly associated with -amyloid levels, senile plaque accumulation and choline acetyltransferase activity in different brain areas. These observations could suggest an involvement of the PON1 in AD pathogenesis and response to treatment.

In the present study it was seen that both investigated kinds of PON1 polymorphism: the -108C>T and the Q192R one exerted a considerable influence on PON1 arylesterase activity. PON1 activity was the high­est in CC and the lowest in TT genotype (CC>CT>TT) of promoter -108C>T polymorphism. The same results were obtained by other authors in population-based studies [3,28]. It was suggested that -108 polymorphism is localized within a probable binding site for Sp1, a ubiquitous transcription factor common in TATA-less genes such as PON1 [3].

We have noticed some differences of PON1 activity depending on Q192R polymorphism though the substrate named by some authors as a “non-discriminating” one was used. The influence of coding p.Q192R polymorphism on enzyme activity was considerably weaker than influence of promoter variants. The highest activity was shown in QQ and the lowest in RR genotype (QQ>QR>RR). The difference was particularly pronounced when the influence of promoter polymorphism was taken into account.

The multivariate regression analysis performed in our study confirmed that PON1 polymorphisms at positions -108 and 192 are the independent determinants of PON1 arylesterase activity. The most prominent contribution is made by the -108 T allele (it explained about 40% of the variance of the PON1 activity both in dementia and controls). A smaller but significant influence of 192R allele on enzyme activity is also observed (2.6% in dementia and 6.1% in controls).

The influence of the p.Q192R polymorphism on PON1 arylesterase activity was also stated by other authors [3,10,23]. One has to admit that this kind of polymorphism does not preclude interference with the activity of the enzyme.

Conclusions

1. Low PON1 activity has a dominant influence on the de­mentia risk.

2. PON1 gene promoter polymorphism plays a consi­derable role in PON1 activity, therefore, it could have an additional role in dementia development, parti­cularly in Alzheimer’s disease. The p.Q192 polymor­phism could have less influence.

3. The results of the haplotype analysis for combination of both PON1 polymorphisms suggest that the rare T-R haplotype including both -108T and 192R alleles could be an ‘at-risk haplotype’ of dementia of the neurodegenerative type (AD and MD).

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