PON1 PCR-RFLP polymorphism frequency and enzyme activity were determined in 223 patients with dementia (94 with AD, 55 with VaD and 74 with MD) and in 100 age and sex matched controls without dementia. I found no statistical significance of genotype frequencies between analyzed groups. &#xd;&#xa;Paraoxonase 1 activity was lower in patients carrying the R allele in locus 192 as compared with the R allele non-carriers. The results showed statistically significant association between PON1 polymorphism and enzyme activity and thus can suggest an important relationship between different isoforms of PON1, enzyme activity and dementia

Tomasz Krzywkowski

English

Nature Precedings

18
th
 March 2010 Bachelor's Degree Project summary 
 
Page 1/2 
 
BEng Tomasz Krzywkowski 
The work was performed  in the Departament of Genetics Institute of Psychiatry and Neurology 
Warsaw, Poland 
Paraoxonase 1 Polymorphism p.Q192R 
in Patients With Dementia 
Abstract 
PON1 PCR-RFLP polymorphism frequency and enzyme activity were determined in 223 patients with 
dementia (94 with AD, 55 with VaD and 74 with MD) and in 100 age and sex matched controls without 
dementia. I found no statistical significance of genotype frequencies between analyzed groups.  
Paraoxonase 1 activity was lower in patients carrying the R allele in locus 192 as compared with the R 
allele non-carriers. The results showed statistically significant association between PON1 polymorphism 
and enzyme activity and thus can suggest an important relationship between different isoforms of PON1, 
enzyme activity and dementia. 
Key words: paraoxonase 1, dementia, polymorphism; 
Abbreviations: HDL-C: high-density lipoprotein (HDL-cholesterol); LDL: low-density lipoprotein; PON: 
paraoxonase; AD: Alzheimer disease; VaD: vascular dementia;  MD: mixed dementia. 
___________________________________________________________________________________________________________________________________
 
1. Introduction 
 Paraoxonase 1, PON1(aryldialkylphosphatase, E.C.3.1.8.1) is a calcium dependent esterase 
present in serum and synthesized by liver. Serum PON1 resides on high-density lipoprotein HDL3  and 
it is known to contribute to antioxidant protection of low-density lipoprotein LDL. By playing  
a protective role against LDL oxidation, PON1 prevents initiation and progression of atherosclerotic 
lesions and thus development of cardiovascular diseases. PON1 exists in many polymorphic forms. 
However, aminoacids 192 or/and 55 substitutions along coding region of PON1 distinguish two most 
abundant isoforms of  PON1. The aim of this work was to investigate PON1 pQ192R polymorphism 
in Polish patients with various types of dementia, and to study its association with PON1 serum 
activity. 
2. Materials and methods 
223 subjects were investigated, 94 with Alzheimer’s disease (25 males and 69 females mean age 
73±8,56); 55 with dementia of vascular origin (27 males and 28 females mean age 72±7,70);  
and 74 with mixed dementia (28 males and 46 females mean age 75±7,45). The controls consisted of 
100 people without dementia (44 males and 56 females mean age 71±7,75). Dementia was recognized 
according to ICD-10 criteria using MMSE test (Minimental State Examination), clock drawing test 
and neuroimaging methods (magnetic resonance, computer tomography). The type of dementia was 
diagnosed basing on NINCDS-ADRDA scale for AD and NINDS-AIREN scale for VD. 
All genotyping was conducted by PCR amplification (with primers described by Humbert et al. 
1993), with 2µl of DMSO, and OptiTaq polymerase (Eurx) and at an annealing temperature of 58°C 
for 35 cycles. The PCR was followed by polymorphism-specific restriction digestion and gel 
electrophoresis. The Q192R polymorphism was detected by AlwI endonuclease (New England 
BioLabs). 20µl of amplified product was digested with 2,5U of enzyme for 40-48 hours at 37°C. 
When arginine is present at 192, two digested band of 33bp and 99 bp are observed. Serum PON1 
activity was determined spectrophotometrically basing on Kitchen et al.(1973) method, using 
phenyloacetate as substrate.  
Statistical analyses were performed using Statistica 8.0. p values lower than 0,05 were 
considered to be statistically significant. HDL3 serum level, MMSE score and PON1 c.-108C>T 
promoter polymorphism in all patients (all three features considered in statistical analysis),  
were determined previously at the department. 
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
11
.6
33
3.
1 
: P
os
te
d 
4 
Se
p 
20
11
Bachelor's Degree Project summary 18th March 2010 
 
Page 2/2 
 
Results 
Table 1. Distribution of PON1 genotype and allele frequencies in patiens with dementia and controls. 
Genotype Allele 
 
 QQ QR RR   Q R  
 
Group 
N  (%) N  (%) N  (%) ∑ N pa N (%) N  (%) pb 
AD 49(48,9) 52,13 38(37,6) 40,43 7(7,5) 7,45 94 0,704 136 72,34 52 27,66 0,410 
MD 43(43,14) 58,11 27(26,72) 36,49 4(4,14) 5,41 74 0,986 113 76,36 35 23,64 0,939 
VD 34(32,07) 61,82 16(19,85) 29,09 5(3,07) 9,09 55 0,589 84 76,36 26 23,64 0,943 
Dementia 126(124,3) 56,50 81(84,38) 36,32 16(14,37) 7,18 223 0,919 333 74,66 113 25,34 0,717 
Controls 58(57,76) 58,00 36(36,48) 36,00 6(5,76) 6,00 100 --- 152 76,00 48 24,00 --- 
Values show in parentheses are expected values calculated from the Hardy-Weinberg equilibrium.  
AD: χ2=0,0378, df=2, p=0,9812; MD: χ2= 0,0081; df= 2, p = 0,9959; VD: χ2= 2,0762, df= 2,  p = 0,3541;  
Dementia overall: χ2= 0,3435, df= 2, p =0,8422; K: χ2= 0,0173, df= 2, p = 0,9914; 
pa: AD,MD or VD vs. controls; pb: AD,MD or VD vs. controls. 
 
According to table 1, no difference was observed in the allele/genotype distribution  
between demented and controls, however frequency of Q allele was higher in both groups. Stepwise 
regression analysis (table 2), revealed statistically relevant association between PON1 192R allele 
possession and PON1 lower enzyme activity. Presented in table 2 model at the level of 32,6% 
(R2=0,326) explains influence of six non-dependent variables on PON1 enzyme activity, where the R 
allele, explains it by 1,43% (∆R2=0,0143).Negative 
β value means negative influence of the variable.  
According to data displayed in table 3, the 
influence of p.Q192R polymorphism on PON1 
enzyme activity is observed. The highest PON1 
activity was in QQ homozygotes, both in patients and 
in controls. In demented QQ homozygotes patients, 
the level of enzyme activity was equal to 149,7 
µmol/min/ml, while the enzyme activity in the 
carriers of the R allele was equal to 138,6 
µmol/min/ml. The 7,25% activity drop was 
statistically relevant after adjusting for age, sex, 
HDL-C concentration and promoter PON1  
-108 CC polymorphism. 
 
3. Discussion 
 Genotype and allele frequencies in Polish 
patients were similar to patients of Ireland, Italy and 
France. It was observed in this work,  
that PON1 enzyme activity was lower 
in R carrier patients. Presented data 
suggest advantageous role of Q allele. 
On the contrary, Pola et al.(2005) 
demonstrated that subjects carrying 
“disadvantageous” R allele, are more likely to 
respond to cholinesterase inhibitors (Alzheimer 
form of treatment). Interestingly, Lescai et 
al.(2009) and Rea et al.(2004) demonstrated, that PON1 192RR homozygotes were mostly abundant 
among Italian centenarians and nonagenarians.  
In conclusion, categorizing a patient to a risk group, basing on his/her PON1 p.Q192R polymorphism 
is currently impossible. A significant influence of environmental factors on PON1 enzyme activity 
suggest that PON1 activity measurement could  be an additional indicator of  vascular and 
neurodegenerative diseases. 
 
 PON1-activity 
regression summary 
Stepwise regression 
summary 
 
Non- 
dependent variables 
 
β# 
 
p 
 
∆R2 
 
p 
PON1 pC-108 CC 0,50 <1*10-6 0,24 <1*10-6 
HDL-CH 0,19 0,0012 0,04 0,0009 
sex  - 0,16 0,0090 0,02 0,0203 
age  - 0,14 0,0204 0,02 0,0271 
MMSE 0,12 0,0396 0,01 0,0619 
PON1 pQ192R-R  - 0,12 0,0409 0,01 0,0409 
R2=0,346; R2 adjusted=0,326; p<0,000001 
 Table 2. Influence of both genetics and non-genetics factors on PON1  
activity (stepwise regression). 
Variables account in analysis:  
PON1 -108 CC (1= homozygous CC genotype of the promoter region,  
0= CT or TT genotype); HDL-C (HDL serum concentration); sex (1=M, 
0=F); age (years); MMSE (MMSE score); PON1 p192 RR+QR (1= R 
carrier, 0= QQ genotype) 
β    -standardized partials of regression coefficient;  
R2  -determination coefficient; ∆R2- determination coefficient alter; 
#     -positive β value means a positive while negative values a negative 
influence of variable on PON1 (female sex, age and R allele result in 
decreasing of enzyme activity). 
Table 3. Influence of allele R in locus 192 on PON1 enzyme activity 
(t-Student test, analysis of covariance in parentheses). 
Values adjusted for age, sex, promoter PON1 -108 CC homozygous 
genotype, HDL-C concentration, age and sex are shown in parentheses. 
 
 
Group 
 
Genotype 
 
N 
 
PON1 
activity(µmol/
min/ml) 
 
Standard 
deviation 
SD 
P 
 t-Student 
test 
(ANCOVA) 
R carrier (RR+QR) 88 140,1 (138,6) 38,07 Dementia 
R non-carrier (QQ) 116 149,7 (149,7) 38,48 
0,1321 
(0,0157) 
R carrier (RR+QR) 37 158,4 (158,0) 40,89 Controls 
R non-carrier (QQ) 52 174,0 (175,1) 42,42 
0,0683 
(0,0157) 
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
11
.6
33
3.
1 
: P
os
te
d 
4 
Se
p 
20
11


Paraoxonase 1 Polymorphism p.Q192R in Patients With Dementia

http://precedings.nature.com/documents/6333/version/1

Paraoxonase 1 Polymorphism p.Q192R in Patients With Dementia

Abstract

Similar works

Full text

Available Versions

Nature Precedings