Atherosclerosis and Lipoproteins |
From the Department of Neurology (R.S., F.F., P.K., G.R., A.L., H-P.H.), the Institute of Medical Biochemistry (H.S., G.M.K.), and the MRI Center (R.S., F.F., P.K.), Karl-Franzens University, Graz, Austria.
Correspondence to Dr Reinhold Schmidt, Department of Neurology, Karl-Franzens University, Auenbruggerplatz 23, A-8036 Graz, Austria. E-mail reinhold.schmidt{at}kfunigraz.ac.at
| Abstract |
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Key Words: white matter lesions cerebral small-vessel disease paraoxonase genetics
| Introduction |
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The strong association of WMLs with aging and a previous study of our own demonstrating an inverse relationship between lesion extent and plasma levels of antioxidants8 suggest that genes involved in oxidative defense could play a role in the etiology of such brain abnormalities. Free-radical formation increases significantly with aging,9 and increased lipid peroxidation and oxidative stress due to excess free-radical activity and impaired antioxidant defenses have been associated not only with large- but also with small-vessel disease,10 11 the most likely cause of WMLs in the elderly.2 Paraoxonase has antioxidative potential12 and could thus protect against both macrovascular and microvascular diseases, even though they represent distinct vascular pathologies. So far, there have been several publications relating paraoxonase to pathological phenotypes of large-vessel disease, such as coronary heart disease13 14 15 and carotid atherosclerosis16 ; a single study found an association with small-vessel diseaserelated diabetic retinopathy.17 The paraoxonase gene is located at q21 to q22 on the long arm of chromosome 7.18 The ability of paraoxonase to detoxify organophosphorus compounds has been known for years, and its enzyme activity had been determined earlier by the use of the pesticide paraoxon. White populations have a triphasic distribution of paraoxonase activity toward paraoxon,19 which is caused by an amino acid substitution at position 191. Glutamine (A allele) is replaced by arginine (B allele) in its high-activity isoform.18 The B allele has been shown to be associated with coronary heart disease.13 14 15 Another frequent polymorphism at position 54 leads to a methionine (M allele)leucine (L allele) interchange.18 The 2 polymorphisms are in linkage disequilibrium with leucine at position 54, giving rise to arginine at position 191.18 We explored whether these genetic variants influence the occurrence and progression of WMLs in a large cohort of randomly selected, community-dwelling middle-aged and elderly individuals who have been followed up over a time period of 3 years.
| Methods |
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Vascular Risk Factors
Historical information and laboratory findings at baseline and
follow-up were considered for diagnosis of arterial
hypertension, diabetes mellitus, and cardiac disease, including
embologenic abnormalities, coronary heart disease, and left
ventricular hypertrophy. We also assessed
smoking habits and body mass index (BMI). Lipid status including the
levels of plasma triglycerides, total
cholesterol, LDL cholesterol, HDL
cholesterol, and Lp(a) lipoprotein, as well as measurement
of plasma fibrinogen, was determined for each study participant
at both examinations. A detailed description of the definitions of risk
factors and of the laboratory methods used has been published
previously.16 The means of systolic and
diastolic blood pressures, fasting blood sugar, BMI, lipid,
and fibrinogen values on baseline and follow-up measurements were
calculated and used for data analyses.
Isolation of DNA and Genotype Analysis
High-molecular-weight DNA was extracted from
peripheral whole blood by using Qiagen genomic tips (Qiagen
Inc) according to the protocol supplied by the manufacturer. Genotyping
of the Met-Leu54 polymorphism was done by polymerase chain reaction
(PCR) amplification of a 170-bp-long fragment and using the primers
described by Humbert et al.18 The PCR products were
cleaved by NlaIII in the presence of BSA at 37°C for 3
hours. The digested products were analyzed on a 15%
polyacrylamide gel, stained with ethidium bromide, and examined
under UV transillumination. The L allele corresponded to
the nondigested 170-bp-long fragment, whereas the M
allele corresponded to 126- and 44-bp fragments. A similar protocol
was used for genotyping the Gln-Arg191 polymorphism and also using
the primers described by Humbert et al.18
Magnetic Resonance Imaging
MRI was performed on 1.5-T superconducting magnets
(Gyroscan S 15 and ACS, Philips) with proton-density and T2-weighted
(repetition time [TR]/echo time [TE], 20002500/3090 ms)
sequences in the transverse orientation. T1-weighted images (TR/TE,
600/30 ms) were generated in the sagittal plane. Slice thickness was
5 mm and the matrix size used was 128x256 pixels. The MRI
protocols at baseline and at the 3-year follow-up were identical. The
scanning plane was always determined by a sagittal and coronal pilot to
ensure consistency in image angulation throughout the
study. The baseline and follow-up scans of each study participant were
read independently by 3 experienced investigators who were blinded to
the clinical data of study participants. Blinding of the readers for
the date of the examinations was impossible because the format of hard
copies had changed from baseline to follow-up. According to our scheme,
WMLs included abnormalities in the subcortical region and deep white
matter as well as irregular periventricular lesions
extending into the deep white matter.3 They were graded
into absent (grade 0), punctate (grade 1), early confluent (grade 2),
and confluent (grade 3) abnormalities.3 The number of WMLs
was recorded and categorized into 0, 1 to 4, 5 to 9, and >9
lesions.
Values for interrater agreement regarding WML grade
at baseline and at 3 years ranged from 0.63 to 0.70 and from 0.66 to
0.68, respectively, in regard to the number of lesions. We disregarded
caps and pencil-thin periventricular linings because they
represent normal anatomic variants.21 22 We also
disregarded a smooth "halo" surrounding the lateral ventricles
because it is nonischemic in etiology, according to
histopathological correlations.21 A change of WMLs in
grade or number from baseline was determined by direct scan comparison.
The final rating of WML progression relied on majority judgment of the
3 assessors. In case of complete disagreement, consensus was found in a
joint reading session. The interrater agreement for WML progression
ranged from 0.58 to 0.68. The Figure
displays examples for each WML grade and
for WML progression. We also recorded lacunes. They were defined as
focal lesions isointense to cerebrospinal fluid and involving the basal
ganglia, the internal capsule, the thalamus, or the brain stem and not
exceeding a diameter of 10 mm.
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Carotid Artery Duplex Scanning
Color-coded equipment (Diasonics, Vingmed CFM 750) was used to
determine vessel wall abnormalities of the carotid arteries in all
participants. The imaging protocol, grading of atherosclerotic changes,
and the associations between duplex findings and paraoxonase
genotypes in our study population have been reported
previously.16 In the current study, we describe the
presence of atherosclerotic changes among the genotype subsets
and adjust for this variable when assessing the influence of the
paraoxonase polymorphisms on WMLs.
Statistical Analysis
We used the Statistical Package for the Social Sciences
(SPSS 8.0) for data analysis. Categorical
variables among the paraoxonase genotypes were compared by
the
2 test. Assumptions of a normal
distribution for continuous variables were compared by Lilliefors
statistics. Normally distributed continuous variables were compared
by 1-way ANOVA, whereas the Kruskal-Wallis test was used for comparison
of nonnormally distributed variables. Allele frequencies were
calculated by the gene counting method, and Hardy-Weinberg equilibrium
was assessed by the
2 test. The relative
contribution of the paraoxonase genotypes to the presence of
WMLs at baseline and to WML progression at the 3-year follow-up was
assessed by multiple logistic regression analysis.
Forward-selection stepwise regression analysis was used to
create a model of independent predictors of MRI findings. At each step,
each variable not yet in the model was assessed for its
contribution to the model, with the most significant variable to be
added. This process continued until no further variable made a
significant (P<0.05) contribution to the model. Odds ratios
(ORs) and 95% confidence intervals (CIs) were calculated from the ß
coefficients and their SEs.
| Results |
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Table 1
compares demographic
variables and risk factors among the LL, LM,
and MM genotype subsets. Individuals with the
MM genotype had slightly higher fasting glucose
levels than did those with the LM or LL
genotype, and there was a nonsignificant trend for lower
triglyceride levels in LM carriers. These
between-group differences diminished further after correction for
antidiabetic and lipid-lowering treatment. There existed no significant
difference among the AA, AB, and BB
genotype subsets when the demographic variables and risk
factors listed in Table 1
were compared (data not shown). Duplex
scanning showed atherosclerotic changes of the carotid arteries in 71
(64.0%) subjects with the LL genotype but in only
56 (47.5%) LM and 18 (51.4%) MM carriers
(P=0.04). Carotid artery abnormalities were also seen in 81
(55.5%) AA, 51 (52.0%) AB, and 13 (65.0%)
BB carriers (P=0.56).
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At baseline, 171 (64.8%) participants had WMLs on MRI. After 3 years,
progression of abnormalities had occurred in 47 (17.8%) subjects.
Regression of abnormalities was never observed by the majority of
readers. A breakdown of WML findings by paraoxonase genotypes
is given in Table 2
. As shown in the
Table
, the PON1 polymorphisms had no significant
influence on baseline results. Yet subjects homozygous for the
L allele at position 54 showed a trend toward higher
grades of WMLs at the initial MRI examination than did their
counterparts with either the LM or MM
genotype. Progression of lesions over 3 years occurred at a
significantly higher frequency in the LL genotype
subset. There was a significant association between WML findings at
baseline and the presence of carotid atherosclerosis
(P=0.017). The association was mainly due to a higher
frequency of grade 2 or 3 WMLs in subjects with carotid changes than in
those with a normal sonographic examination (5.9% versus 16.6%).
Progression of WMLs was not related to the presence of carotid
atherosclerosis. At baseline, lacunes were found in a
total of 17 subjects and were always seen in the basal
ganglionic/thalamic region. At the 3-year follow-up, new lacunes had
evolved in 8 subjects. There was no significant difference in the
distribution of subjects with lacunes at baseline and with evolving
lacunes at follow-up among the genotypes, but the numbers in
the comparative subsets were small.
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Logistic regression analysis yielded an unadjusted OR of 2.38
(95% CI, 1.25 to 4.53; P=0.008) for progression of WMLs in
the LL genotype relative to the 2 other
genotypes. The OR after adjustment for age and sex was 2.55
(95% CI, 1.32 to 4.96; P=0.005). Evaluation for the effect
of the Gln-Arg191 polymorphism demonstrated that WML progression
occurred more commonly in BB carriers, but these differences
with respect to the AA and BB genotypes
were nonsignificant. The Gln-Arg191 polymorphism did not modulate
the effect of the LL genotype on WML progression
because lesion progression was seen at almost identical frequency in
11(23.9%) subjects in the LL/AA group and in 5
(26.3%) individuals in the LL/BB group. When we
used forward stepwise regression analysis to create a model of
predictors of WML progression, the LL genotype
remained in this model in addition to age and diastolic
blood pressure. Age entered the model first, the LL
genotype second, and diastolic blood pressure third
(Table 3
). All other variables,
including sex, BMI, systolic blood pressure, fasting glucose
level, diabetes, smoking status, cardiac disease, blood lipids, plasma
fibrinogen level, and carotid atherosclerosis did not
enter the model. When carotid atherosclerosis was
forced into the model, the OR for the association between the
LL genotype and WML progression did not materially
change.
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| Discussion |
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In line with these results, Garin et al25 found that the Leu-Met54 polymorphism is of central importance to paraoxonase function because it influences the serum activity and concentration of the enzyme, whereas the 191 variant has only little effect. This finding, together with the inconsistent results of other studies on the association between the Gln-Arg 191polymorphism and coronary heart disease in genetically distinct populations,26 suggests that the PON1 polymorphism at position 191 is not causal but rather may be in linkage disequilibrium with a functional sequence variant in the vicinity. Whether the Leu-Met54 polymorphism represents this variant cannot be elucidated in allelic association studies. PON1 belongs to a multigene family including PON2 and PON3 at the same locus on chromosome 7.27 Two polymorphisms in the PON2 gene have only recently been described. Their functional importance is not yet fully determined, but like the PON1 polymorphisms, they were linked to coronary heart disease.28
Several studies support the role of paraoxonase in atherogenesis. The enzyme is linked to HDL and may be partly responsible for the antioxidative effect of this lipid fraction.12 19 Paraoxonase decreases lipid peroxide accumulation on LDL,29 a process that takes place in the subendothelial space.30 In line with this presumed location of action, paraoxonase was found to be present in interstitial fluid in an enzymatically active form.31 Results in PON1 knockout mice have demonstrated that HDL from PON1-deficient mice is unable to prevent LDL oxidation in a coculture model of the arterial wall.32 Paraoxonase immunoreactivity is seen in atherosclerotic lesions, and its intensity increases with their progression.33 There exists much less information on the association between paraoxonase and microvascular disease, which is the most likely cause of WMLs.2 Yet a study of Kao et al17 investigated the role of PON1 polymorphisms in small-vessel diseaserelated diabetic retinopathy and found that the LL genotype at position 54 was associated with this condition, whereas there existed no effect of the Gln-Arg191 polymorphism. These results are consistent with our findings on WMLs. Importantly, in the context of our study results, Primo-Parmo et al27 reported PON1 expression in adult mouse brain and described sequence homologues isolated from a postnatal human brain cDNA library.
Conceivably, the functional significance of the PON1 polymorphism at position 54 is due to its effect on enzyme activity and concentration caused by altered gene expression.34 Unfortunately, we have no frozen serum from our study participants and therefore could not measure these variables in our study group.
In summary, our data suggest a moderate modulatory effect of the Leu-Met54 polymorphism of the PON1 gene on the extent and progression of small-vessel diseaserelated MRI WMLs. Identification of individuals at risk for an unfavorable evolution of these brain changes is important, because increasing lesion load commonly results in cognitive decline and other neurological signs and symptoms such as gait disturbances and a tendency to falls.1 35
| Acknowledgments |
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Received September 27, 1999; accepted January 20, 2000.
| References |
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