Abstract
Background: Although the APOE ε4 allele is a well-known risk factor for developing AD, the impact of the ε4 allele on clinical manifestations in patients with AD is still controversial. One possible reason for this controversy is that previous studies did not consider the effect of patient age at symptom onset.
Objective: To investigate the possible impact of patient age at onset of AD on the effect of APOE genotype on regional cerebral glucose metabolism (rCMRglc).
Methods: The authors compared rCMRglc between probable AD patients (based on criteria of the National Institute of Neurologic Disease and Stroke/AD and Related Disorders Association) with APOE ε4/4 and APOE ε3/3 alleles in early-onset (≤65 years old) and late-onset (>65 years old) groups. In each group, the patients with APOE ε4/4 and APOE ε3/3 alleles were comparable for age at onset, age at examination, sex, disease duration, education level, and severity of dementia.
Results: In the early-onset group, the patients with the APOE ε4/4 genotype showed a significant decrease of rCMRglc in the medial temporal lobe and a significant increase of rCMRglc in the inferior parietal and posterior temporal cortices as compared with those patients with the APOE ε3/3 genotype. In the late-onset group, there were no significant differences in the rCMRglc pattern between the patients with APOE ε4/4 and APOE ε3/3 alleles.
Conclusions: The current findings indicate that the impact of the APOE ε4 genotype on cerebral glucose metabolism of patients with AD may be a function of age at symptom onset.
The APOE ε4 allele is a well-known risk factor for developing AD, and it lowers the age at onset in a dose-dependent fashion.1-4⇓⇓⇓ The impact of the APOE ε4 allele on clinical expression in AD is still debated, however. Although studies with PET have indicated the emergence of parietal glucose hypometabolism before the development of substantial cognitive impairment in subjects carrying the APOE ε4 allele,5,6⇓ the effects of the APOE ε4 allele on regional cerebral perfusion and metabolism in clinical AD patients are still undetermined. Two studies with SPECT demonstrated that an increased number of APOE ε4 alleles is associated with decreased cerebral perfusion in the left occipital cortex7 and bilateral parietal and occipital cortices8 in clinical AD patients. Conversely, two PET studies reported that the APOE ε4 allele was related to relatively preserved regional cerebral glucose metabolism (rCMRglc) in the frontal9 and frontotemporoparietal association cortices.10 Greater parietal perfusion asymmetry has been also associated with the absence of the APOE ε4 allele in AD patients.11 Other PET studies failed to demonstrate any significant differences in cerebral glucose metabolism between AD patients with and without the APOE ε4 allele.12,13⇓
With respect to the relation between the APOE ε4 allele and other clinical manifestations, including cognitive function and brain atrophy in AD patients, the reported results are also controversial. Some studies have reported that the APOE ε4 allele is associated with poorer memory function14,15⇓ and relatively preserved verbal15,16⇓ and visuocognitive function,16 although others have failed to demonstrate such differences.17-19⇓⇓ By using MRI, it has been reported that atrophic changes in the medial temporal lobe structures are more severe in AD patients carrying the ε4 allele,7,14,16,20,21⇓⇓⇓⇓ although other volumetric studies failed to demonstrate a significant difference in the hippocampal volume between AD patients with the APOE ε4 allele and those without it.22-24⇓⇓ A reverse effect of the APOE ε4 allele on whole-brain atrophy16,25⇓ and frontal lobe atrophy20 was also reported.
One possible explanation of these conflicting findings might be interaction between the effect of age at onset of dementia and the clinical manifestations of AD. The age at onset of symptoms is one of the most commonly proposed bases for subtyping AD. Although results are not always consistent, early-onset AD patients are reported to show greater language and/or visuocognitive disturbances26-31⇓⇓⇓⇓⇓ and less memory impairment,32 more severe global cerebral atrophy,33,34⇓ lower rCMRglc in the frontoparietotem-poral cortices,35-39⇓⇓⇓⇓ and higher rCMRglc in the limbic/ paralimbic area39 as compared to late-onset AD patients. Asymmetries of cortical glucose metabolism are also reported to be observed in early-onset AD.40 As described previously, these cognitive, morphologic, and functional differences between AD patients with early and late onset of dementia resemble those reported between AD patients with and without the APOE ε4 allele.
The APOE ε4 allele is considered to lower the age at onset of AD.1-4⇓⇓⇓ The presence of the APOE ε4 allele makes the late-onset type of AD occur earlier; thus, the late-onset type of AD could occur during the “early-onset period” (before the age of 65 years) in some patients. If this is true, we can expect that in patients who have early-onset AD, some of those with the APOE ε4 allele actually have the late-onset type of AD (figure 1 ⇓). Therefore, the differences between AD patients with and without the APOE ε4 allele that have been observed in previous studies may actually be a result of the differences between AD patients with early and late onset of dementia. Consistent with this hypothesis, studies that reported a significant association between the APOE ε4 allele and cerebral glucose metabolism,9,10⇓ brain atrophy,7,14,16,20,21⇓⇓⇓⇓ or cognitive function14,15⇓ examined cohorts that consisted of relatively younger patients. To test this hypothesis, we examined prospectively collected records of the clinical data, PET imaging with 18F-fluorodeoxyglucose (FDG), and APOE genotype of AD patients admitted to our hospital. We evaluated the effect of the APOE ε4 allele on cerebral glucose metabolism, classifying AD cases into early-onset and late-onset subtypes. Because a previous study found no difference in the pattern of rCMRglc between AD patients with APOE ε3/4 and APOE ε3/3 alleles,12 we compared APOE ε4 homozygotes with APOE ε3 homozygotes in the present study so as to maximize the contrast.
Figure 1. Hypothesis: because the presence of the APOE ε4 allele makes late-onset type AD occur earlier, some patients with the late-onset type of AD could have the onset of AD before the age of 65 years. When patients who have the onset of AD during the “early-onset period” are studied, we may find the same differences between the patients with and without the APOE ε4 allele as the differences found between the early-onset and late-onset subtypes, because there are more patients with the late-onset type of AD in the patients with the APOE ε4 allele than in the patients without the APOE ε4 allele.
Figure 2. Results of the Statistical Parametric Mapping 99 (Wellcome Department of Neurology, London, UK) analyses. PET statistical maps were coregistered with MRI scans. Left column shows the area where glucose metabolism is significantly decreased; right column shows the area where glucose metabolism is significantly increased in patients with the APOE ε4/4 allele as compared with patients with the APOE ε3/3 allele. SPM = statistical parametric mapping.
Methods.
This study was conducted at Hyogo Institute for Aging Brain and Cognitive Disorders (HI-ABCD), a research-oriented hospital for dementia. All procedures followed the 1993 HI-ABCD Ethical Committee’s Clinical Study Guidelines and PET Drug Usage Manual and were approved by the Institutional Review Board. Written informed consent was obtained from all patients and their caregivers according to the 1975 Helsinki Declaration of Human Rights.
Subjects.
From the database of the HI-ABCD Dementia Registry, we selected AD patients (fulfilling the criteria of the National Institute of Neurologic Disease and Stroke/AD and Related Disorders Association for probable AD41) who had either the APOE ε3/3 or APOE ε4/4 allele and were studied for cerebral glucose metabolism by PET between May 1995 and March 2001.42 During this period, the clinical and investigative data for the patients who were admitted to the HI-ABCD infirmary on a short-term basis for examination of dementia were collected prospectively in a standardized fashion and entered into the registry. The database includes findings of examinations by neurologists and psychiatrists, routine laboratory tests, standard neuropsychological examinations, EEG, MRI of the brain, MR angiography of the neck and head, and cerebral perfusion/metabolism studies by PET or SPECT. Patients were excluded because of the following: 1) complications of other medical illnesses possibly causing cognitive impairment, including thyroid disease, vitamin deficiencies, and malignant disease; 2) complications of developmental abnormalities, mental disease, substance abuse, or significant neurologic antecedents such as brain trauma, brain tumors, epilepsy, and inflammatory disease; 3) evidence of focal brain lesions on MRI, including lacunar infarcts and hematomas; 4) suggestion of autosomal dominant transmission; 5) left handedness or ambidexterity; or 6) lack of informed consent.
There were 87 patients who fulfilled the criteria. Among them, we selected 57 patients for the present study by the following procedures. First, we divided these 87 patients into a group of 41 patients whose onset of symptoms had occurred at the age of 65 years or younger and a group of 46 patients whose onset of symptoms had occurred at the age of 66 years or older. The age at symptom onset was defined as the age at the first appearance of symptoms of sufficient severity to interfere with social or occupational functioning.43 There were 32 APOE ε3 homozygotes (mean age at onset: 55.8 ± 4.5 years, 19 women and five men) and nine APOE ε4 homozygotes (mean age at onset: 61.3 ± 5.8 years, five women and four men) in the patients with early-onset disease and 36 APOE ε3 homozygotes (mean age at onset: 73.3 ± 4.8 years, 29 women and 7 men) and 10 APOE ε4 homozygotes (mean age at onset: 70.9 ± 4.3 years, six women and four men) in the patients with late-onset disease. Second, because gender proportion and distribution of age at onset were significantly different between APOE ε3 and APOE ε4 homozygotes in each age at onset group, we made up sex- and age-comparable groups as follows. For the early-onset group, we selected the 10 oldest women at onset and the eight oldest men at onset from 32 APOE ε3 homozygotes with early-onset disease, so that 18 APOE ε3 homozygotes and nine APOE ε4 homozygotes were sampled (2:1 ratio). For the late-onset group, we selected the 13 youngest women at onset and all seven men from 36 APOE ε3 homozygotes with late-onset disease. A total of 20 APOE ε3 homozygotes and 10 APOE ε4 homozygotes were sampled (2:1 ratio). Although other clinical variables such as educational level, disease duration, and disease severity are likely to affect cerebral metabolism, the education level, disease duration, and severity of cognitive disturbances, as shown by their Mini-Mental State Examination scores, were comparable between APOE ε3 homozygotes and APOE ε4 homozygotes in each age at onset group (table 1⇓).44
Clinical characteristics of the patients with APOE ε4/4 and with APOE ε3/3 alleles
Determination of APOE genotype.
The detailed method for APOE genotyping is described elsewhere.45 In brief, genomic DNA was extracted from peripheral blood with a Genomix DNA extraction kit (Talent Corporation, Trieste, Italy) according to the manufacturer’s protocol. The APOE genotype was determined by using PCR restriction fragment length polymorphism according to the procedure described by Wenham et al.46
PET procedure.
Before PET scans, all subjects received MR examinations for diagnosis and PET positioning. The detailed PET scanning procedure for measuring cerebral glucose metabolism is described elsewhere.47 In brief, PET images were obtained with a tomograph (Headtome IV; Shimadzu Corporation, Kyoto, Japan).48 A transmission scan was performed using a 68Ga/68Ge pin source for attenuation correction after each subject was positioned. PET studies were performed with the subjects under resting conditions with their eyes closed and ears unplugged. All subjects had fasted for at least 4 hours before PET scanning. FDG (185–346 MBq) was injected into the right antecubital vein. Brain scanning was started 60 minutes after the injection, and emission data were collected for 12 minutes.
Data analysis.
PET data were directly transmitted to a UNIX workstation (Indigo2 High Impact; Silicon Graphics, Mountainview, CA) from the PET imaging units for file format conversion using Dr. View version 5.2 (Asahikasei Joho System, Tokyo, Japan). The data were then transferred to a Windows NT (Microsoft, Redmond, WA) workstation, on which image analysis was performed.
Voxel-by-voxel statistical analysis processing was performed with Statistical Parametric Mapping 99 (SPM99) software (Wellcome Department of Neurology, London, UK). Calculations and image matrix manipulations were performed with MATLAB (MathWorks, Natick, MA). Each subject’s original FDG image was automatically transferred with SPM99, which uses linear and quadratic parameters to minimize the sum of squares between each subject’s FDG image and template PET image (the H215O image rather than the FDG image), and transformed using these parameters. Anatomically normalized FDG images were then smoothed with an isotropic Gaussian filter (12 mm full width at half maximum), and individual global counts were normalized by proportional scaling to a mean value of 50 mg per 100 mL per minute. Finally, between-group comparisons were performed on a voxel-by-voxel basis on all voxels common to all the subjects.49 The thresholds were set at p = 0.001. Correction for multiple comparisons was not performed.
In addition to the SPM99 analysis, we also used the region of interest (ROI) analyses. For ROI analyses, MRI scans of three-dimensional scales and coordinates identical to PET scans were made for anatomic reference to the PET scans. The PET and MRI scans were displayed side by side on a display monitor together with the stereotaxic atlas of Talairach and Tournoux.50 One or two circular ROIs with a diameter of 10 mm were placed on the regions where significant differences were shown on SPM99 analysis in both hemispheres. To increase reliability, the values of the ROIs in each brain region were averaged. One neuroradiologist blind to the patients’ status conducted ROI placement and measurements. To remove the intersubject difference in baseline metabolism, analyses were based on the normalized value of rCMRglc; in other words, they were based on the ratio of the value of rCMRglc to the mean value of metabolic rates for glucose of bilateral primary sensorimotor cortices, where the histologic changes are largely unaffected in AD.51 We used two-way analysis of variance with one between-subjects factor (APOE genotype) and one within-subjects factor (laterality) and the post hoc Scheffé test for the ROI analyses; the significance level was set at p < 0.05.
Results.
On the SPM99 analysis for early-onset AD, relative glucose metabolism was significantly lower in the right medial temporal lobe, right basal frontal cortex, bilateral basal ganglia, bilateral postcentral gyri, and right medial occipital cortex in the APOE ε4 homozygotes than in the APOE ε3 homozygotes. Conversely, relative glucose metabolism was significantly higher in the left posterior temporal cortex and left inferior parietal cortex in the APOE ε4 homozygotes than in the APOE ε3 homozygotes (see table 2, figure 2). For late-onset AD, no significant differences in rCMRglc were noted between the APOE ε4 and APOE ε3 homozygotes at the threshold (p = 0.001). Even when we reset the threshold at p = 0.01, we could not find significant differences; however, when we reset the threshold at p = 0.05, the differences were apparent in the medial temporal lobe, basal frontal cortex, posterior temporal cortex, and inferior parietal cortex.
The Talairach Tournoux Atlas location of brain regions’ peak significance for the statistical map shown in figure 2
We also compared rCMRglc in the medial temporal lobe, right basal frontal cortex, posterior temporal cortex, and inferior parietal cortex by the ROI analysis for the early-onset AD patients (figure 3, table 3). On two-way analysis of variance, the effect of APOE genotype was significant, but the laterality effect and interaction term were not significant for the posterior temporal and inferior parietal cortices. The post hoc Scheffé test showed that the normalized glucose metabolism was higher in the APOE ε4 homozygotes than in the APOE ε3 homozygotes in these areas. For the normalized glucose metabolism in the medial temporal lobe, we found a significant APOE genotype effect and a significant laterality effect. The interaction term was not significant. The post hoc Scheffé test showed that the normalized glucose metabolism was lower in the APOE ε4 homozygotes than in the APOE ε3 homozygotes and was lower on the right side than on the left side. No significant effects were found in the normalized glucose metabolism in the basal frontal cortex.
Figure 3. Region of interest (ROI) placement. Template MRI scans of brain slices used for ROI analysis. These slices were −13, −6.5, 0, and 32.5 mm above the anterior commissure–posterior commissure line. The ROI for this study include the following areas: 1 = medial temporal lobes; 2 = basal prefrontal cortices; 3 = posterior temporal cortices; 4 = inferior parietal lobules. To increase reliability, the values of ROI in each cortical region were averaged.
Normalized cerebral glucose metabolite rate by the bilateral primary sensorimotor cortices in the early-onset patients with APOE ε4/4 and APOE ε3/3 on region of interest analysis
Discussion.
The current study demonstrated that the pattern of rCMRglc was different between the APOE ε4 homozygotes and APOE ε3 homozygotes in early-onset AD. In late-onset AD, there was no significant difference in the pattern of rCMRglc between the APOE ε4 homozygotes and APOE ε3 homozygotes, although we noted a subtle effect of gene status similar to that found in early-onset AD. In this study, sex and age at onset were well controlled, and background factors were comparable. Therefore, the differences in the pattern of rCMRglc would not be attributable to other variables such as actual age and severity of dementia but to the APOE ε4 genotype.
The APOE ε4 genotype was related to pronounced hypometabolism in the medial temporal lobe and relatively preserved metabolism in the inferior parietal and posterior temporal cortices in early-onset AD. The pattern of the rCMRglc observed in the early-onset APOE ε4 homozygote patients is similar to that recognized in patients with late-onset AD.35-39⇓⇓⇓⇓ Therefore, as hypothesized, the results suggested that the APOE ε4 allele makes late-onset type AD occur earlier. Such a shift in the prevalence from the early-onset type of AD to the late-onset type of AD would not occur at the cutoff age of 65 years. The crossover might explain the subtle effect of the APOE gene status on the rCMRglc pattern. In an MRI volumetric study in AD patients, a significantly smaller hippocampal volume has been associated with the APOE ε4 allele in the younger patients (65–75 years old) but not in the older patients.21 Our results are consistent with the findings of this study.
The SPM99 analysis also demonstrated lower relative glucose metabolism in the bilateral basal ganglia, bilateral postcentral gyri, and right medial occipital cortex in the APOE ε4 homozygotes than in the APOE ε3 homozygotes. In AD, these regions are usually histologically unaffected, however.51 Because glucose metabolism in these regions has been reported to be largely unaffected,52 an interpretation of a transneural hypometabolism is unlikely. Therefore, the findings in these regions were likely epiphenomena occurring as a result of overcorrection of intersubject variance of global cerebral metabolism.
Because the effect of the APOE ε4 allele on the pattern of rCMRglu was significant only in the early-onset patients, the effect would be obscured if we examined the early-onset and late-onset patients together. Failure to demonstrate an effect of the APOE ε4 allele on cerebral glucose metabolism in previous PET studies might be attributable to overlooking the age at onset of AD. Actually, in our previous study,13 which examined patients whose mean age was 69.7 years, we failed to demonstrate a significant correlation of glucose metabolic rate with the number of APOE ε4 alleles in any brain region, although we noted that there was a slight tendency for glucose metabolic rates in the frontal and parietal association cortices to increase and a slight tendency for medial temporal glucose metabolic rates to decrease with an increasing number of APOE ε4 alleles. It is noteworthy that two PET studies9,10⇓ that found a significant relation between the presence of the APOE ε4 allele and relatively preserved rCMRglc in the association cortices examined younger patients.
The generalizability of this study is limited by the small sample size and the retrospective case-controlled study design. Because APOE ε4 alleles decrease the age at onset, the distribution of age at onset is suspected to differ between AD patients with and without APOE ε4 alleles in the general population. Only a case-controlled study on the basis of age at onset can eliminate the effect of age at onset. Another limitation of the present study is the failure to examine the gene dose effect. Because previous studies demonstrated a gene dose effect,1-4⇓⇓⇓ such an effect would be expected in the patients selected for this study.
In conclusion, the impact of the APOE ε4 genotype on clinical manifestations of AD patients may be a function of age at symptom onset. The APOE ε4 genotype is clearly associated with pronounced hypometabolism in the medial temporal lobe and relatively preserved metabolism in the parietotemporal cortices in early-onset AD. The age at onset of dementia should be taken into consideration in future studies on this topic.
- Received July 20, 2001.
- Accepted November 16, 2001.
References
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