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Apolipoprotein E genotype and its pathological correlation in Chinese Alzheimer's disease with late onset
Apolipoprotein E Genotype and Its Pathological Correlation in Chinese Alzheimer's Disease With Late Onset LAN CHEN, MB, LARRY BAUM, PHD, HO-KEUNG NG, MD, FRCPATH, FRCPA, LISA Y,S, CHAN, PHD, AND CHI-PUI PANG, PHD In this study, we attempted to find a relationship between apoliprotein E (ApoE) genotypes and Alzheimer's disease (AD) pathology in different areas of the brain in Chinese. We also studied the borderline group of possible AD (Poss). There were 34 definite or probable AD (Ad), 18 Poss, and 123 brains from age-matched normal subjects (N). ApoE genotype was determined by nested polymerase chain reaction on genomic DNA extracted from archival paraffinembedded materials. Hippocampus (including eutorhinal cortex), amygdala, superior temporal lobe, middle frontal gyrus, and inferior parietal lobule of the brains of Ad and Poss were examined with 13 amyloid (A13) immunostaining, and the same regions plus medial occipital lobe were examined with tau immunostaining. The percentage of plaque area stained for A13 in each brain region was obtained by an image analyzer, and the average number of neurofibrillary tangles stained for tau was counted with an eyepiece graticule. ApoE ¢4 frequency was increased in both Ad (22.1%, X2, df= 1, P = .00005), and Poss (33.3%, P = .000005) compared with N (5.3%). A13 load was
significantly increased in the neocortex in Ad examined with at least 1 copy of ~4 compare d with subjects without ~4 (Mann-Whitney, P = .014). The same trend, though not statistically significant, occurred in Poss (P = .15). Tan expression was associated with ApoE ~4 in neither Ad nor Poss. Poss is genetically and histologically similar to Ad, although the overall A[~ load is significantly increased in the latter. These findings support the recent Consensus Report's findings that all Alzheimer-type pathology may be significant. HUM PATUOL30:11721177. Copyright © 1999 byW.B. Saunders Company Key words:Alzheimer's disease, 13-amyloid plaque, neurofibriliary tangle, apolipoprotein E, possible AD. Abbreviations: AD, Alzheimer's disease; CERAD, the Consortium to Establish a Registry for Alzheimer's Disease; ApoE, apolipoprotein E; A13, 13 amyloid; NFI', neurofibrillary tangle; Ad, definite or probable Alzheimer's disease; Poss, possible Alzheimer's disease; N, normal controls; PAP, peroxidase anti-peroxidase; PCR, polymerase chain reaction; OR, odds ratio; CI, 95% confidence intervals.
A diagnosis of definite Alzheimer's disease (AD) requires confirmation by histology and autopsy even though the clinical diagnosis for AD generally exceeds 80% accuracy) Although histological criteria remain imperfect, neuropathologists involved in the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) established simple and practical neuropathological criteria for AD in 1991 z and updated them in 19933 and 1997. 4 Criteria for levels of AD diagnosis from definite, probable, to possible AD are provided, and they d e p e n d on the presence or absence of a history of dementia and the severity of neuritic plaques in relation to age. In a general hospital where not all elderly patients are assessed carefully as to their mental status and subtle mental deterioration may be overlooked, autopsy brains that demonstrate significant Alzheimertype pathology may be categorized as possible AD (Poss). Currently, there have been few studies on these possible AD cases. Although the apolipoprotein E (ApoE) e4 allele is known to be a strong risk factor for AD, how its presence affects the development of AD is still uncertain. ApoE
has a high avidity to [3 amyloid (A~), and it is found in neuritic plaques and some neurons. It is hypothesized that ApoE4 might be involved in AD pathogenesis by its effects on amyloid aggregation in neuritic plaques or formation of paired helical filaments in the neurofibrillary tangle (NFT). 5 A[3 plaque deposits and NFTs have been observed to be increased in brains of AD patients with ~4 compared with patients without e4. 6,7 However, the association of ApoE e4 with AD pathology is still controversial according to other authors 8-12and deserves further study. Studies suggest that there might be a difference in AD between Chinese and Western populations. AD accounts for approximately 4% of the population older than age 70 years in H o n g Kong is and 3% older than 65 years in Shanghai. 14 These figures are at the lower margin of the prevalence reported in Americans ]~ (10 % over the age of 65) and Europeans TM (3% to 11% over the age of 60). Alzheimer-type change in brains from n o n d e m e n t e d elderly has also been found to be less than that in Caucasians. 17,1s However, there have been no studies correlating different ApoE genotypes with postmortem AD changes in Chinese. In this study, we attempted to perform ApoE genotyping and examine its relationship to A[3 plaque deposits and NFT formation in different areas of autopsy brains in Chinese late onset AD. We also tried to delineate the ApoE genetics and the pathological characteristics of Poss, which to our knowledge has not previously been performed in Chinese.
From the Department of Anatomical and Cellular Pathology, Department of Ophthalmology and Visual Sciences, Department of Chemical Pathology, Chinese University of Hong Kong, Shatin, Hong Kong. Accepted for publication May 12, 1999. Address correspondence and reprint requests to Ho-keung Ng, MD, FRCPath, FRCPA, Department of Anatomical & Cellular Pathology, Prince of Wales Hospital, Shatin, Hong Kong. Copyright © 1999 by W.B. Saunders Company 0046-8177/99/3010-0009510.00/0
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LATE-ONSETALZHEIMER'SDISEASE(Chen et al) MATERIALS AND METHODS
Subjects Thirty-four autopsy brains of definite or probable AD (Ad) with mean age of 82.8 + 7.9 years (standard deviation), 18 cases of Poss having no clinical history of dementia with mean age 78.7 -+ 9.6, and 123 normal controls (N) with mean age 82.0 +- 6.6 were collected in our hospital during 1992 through 1997. Diagnosis was previously made based on CERAD criteria by Bielschowsky and Bodian staining of sections from hippocampus (including entorhinal cortex), amygdala, superio r temporal lobe, middle frontal gyrus, and inferior parietal lobule, s and medial occipital lobe according to Braak and Braak} 9
AI~ and Tau Immunostaining of AD Patients Paraffin sections were cut at a thickness of 4 pm for immunostaining. In each AD case, sections from 1 hippocampus (including entorhinal cortex), amygdala, superior temporal gyrus, middle frontal gyrus, and inferior parietal lobule were stained with a monoclonal antibody against A[3 (DAKO, 1/50) by the avidin-biotin complex method and counterstained by methyl green. The above regions plus sections from 1 medial occipital lobe were stained with a polyclonal antibody against tau for NFTs (DAKO, 1/600, with overnight incubation) by the peroxidase anti-peroxidase (PAP) method. Cases of definite AD were used as positive controls. For negative control, the primary antibody was replaced by 5% normal rabbit serum in the ABC m e t h o d and 2% normal swine serum in the PAP method.
72°C for 5 minutes. One microliter of a 50-fold-dilution of the first PCR product was used for a second PCR by P2 and P3 (Gibco-BRL) in a final volmne of 30 pL with the conditions described, except that 35 cycles of 30 seconds at 95°C, 1 minute at 68°C, and 1 minute at 72°C were used. An aliquot of 25 pL of the second PCR products was digested by 2 U of Hha I (Gibco-BRL) overnight, followed by electrophoresis on 12% polyacrylamide gels as described21 The fragments were visualized by ultraviolet light after staining with ethidium bromide. All cases were successfully genotyped.
StatisticalCalculations For ApoE genotyping, chi-squares, odds ratios (OR), and 95% confidence intervals (CI) were calculated using the Epi6 computer program (Centers for Disease Control, Atlanta, GA). Yates-corrected P values were used unless the expected values were less than 5, when Fisher's exact p values were used instead. Nonparametric tests were used for pathological data analysis using Statistica software (Statsoft, Tulsa, OK).
RESULTS ApoE Polymorphism
A[~ plaque deposits were determined by calculating the percentage area covered by A[Mmmunostained plaques at a magnification of ×100 by using the GAS 200-cell analysis system (Becton Dickinson, Franklin Lakes, NJ). In each of the 5 brain regions mentioned above, 15 to 20 microscopic fields with the most numerous A[3 plaques were counted, and the percentage area occupied by A[3 staining was averaged.
A p o E e4 was significantly i n c r e a s e d in A d o r Poss versus N (X2, d f = 1, P = .00005 f o r Ad; P = .000005 f o r Poss), with a n o d d s r a t i o f o r cases with at least 1 c o p y o f t h e e4 allele c o m p a r e d with cases w i t h o u t ~4 o f 5.9 times f o r AD a n d 10.6 t i m e s f o r Poss (Table 1, 2). As t h e least c o m m o n allele in C h i n e s e , e4 was f o u n d in 5.3% o f c o n t r o l s in this study (Table 2). e2, t h e least c o m m o n allele in whites, 22 was m o r e c o m m o n t h a n ~4 in N in this study. However, it was n o t u n d e r r e p r e s e n t e d in A d o r Poss cases as c o m p a r e d with N by X2 ( d f = 1, P = .77 for Ad, P = 1.00 f o r Poss) a n d o d d s r a t i o analysis (Table 2). A d a n d Poss d i d n o t significantly differ f r o m e a c h o t h e r in t h e d i s t r i b u t i o n o f t h e 3 A p o E alleles (X2, d f = 2, P = .38).
Counting of NFTs
A!3 Deposition in AD Brains
NFTs were counted with an eyepiece graticule at 100× magnification of a Nikon Labophot 2 microscope. In each of the 6 brain regions stained for tau, 15 to 20 microscopic fields of 1 mm 2 containing the most numerous NFTs were counted and an average obtained as NFT density.
I n b o t h A d a n d Poss, A[3 d e p o s i t i o n q u a n t i f i e d as p e r c e n t a g e a r e a was s t u d i e d in 3 r e g i o n s o f n e o c o r t e x , h i p p o c a m p u s , a n d a m y g d a l a f o r e a c h case. T h e r e was n o s i g n i f i c a n t d i f f e r e n c e o f A[3 l o a d a m o n g t h e f r o n t a l , t e m p o r a l , a n d p a r i e t a l cortices in A d o r Poss ( F r i e d m a n ANOVA, P < .54 for A d a n d P < .18 f o r Poss), i n d i c a t i n g t h a t A[3 d e p o s i t s were evenly d i s t r i b u t e d in t h e 3 r e g i o n s o f n e o c o r t e x (Fig 1). T h e r e f o r e , in e a c h case, A[3 l o a d s in t h e 3 r e g i o n s w e r e a v e r a g e d as " n e o c o r t e x " to c o m p a r e with t h a t in h i p p o c a m p u s a n d a m y g d a l a .
Quantification of AI3 Plaque Deposits
ApoE Genotyping For each case, DNA was extracted from 5 pieces of 10-1Jm paraffin tissue sections by digestion with proteinase K (BULK, 10 m g / m L , overnight at 37°C), p h e n o l / c h l o r o f o r m treatment, and precipitation with cold ethanol as described, z° The fourth exon of the ApoE gene was amplified by the two-step nested polymerase chain reaction (PCR) using primers as previously described. 21 The first PCR by primers P1 and P4 (Gibco-BRL), contains about 500 ng genomic DNA, 200 p m o l / L of each dNTE 0.5 lamol/L of each primer, 3.0 m m o l / L magnesium chloride, 10% dimethylsulfoxide, 0.004% gelatin, and 1 U Taq DNA polymerase in a final volume of 20 IlL. After initial denaturing at 95°C for 5 minutes, a touchdown PCR program was carried out for 40 cycles of 30 seconds at 95°C, 30 seconds at 72°C, decreasing 0.4°C per cycle and 1 minutes at 72°C, followed by a final extension at
TABLE 1. ApoEAllele and Genotype Distribution Alleles e3 (%)
e2 (%)
Oenotype e4 (%)
AD 47 (69.1) 6 (8.8) 15 (22.1) Poss 20 (55.6) 4 (11.1) 12 (33.3) N 206 (83.7) 27 (11.0) 13 (5.3)
2/2 2/3 3/3 2/4 3/4 4/4 1 0 3
4 3 21
15 5 86
0 1 0
13 7 13
1 2 0
Abbreviations: AD, Mzheimer's disease; Poss, possible Alzheimer's disease; N, normal controls.
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HUMAN PATHOLOGY
Volume 30, No. 10 (October 1999)
TABLE 2.
Odds Ratios for Alzheimer's Disease in Association With G e n o t y p e s With or Without ApoE e4 Alleles
AD n
Poss
OR
Copies of e4 0 20 1 or 2 14 Copies of e2 0 29 1 or 2 5
p
n
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OR
p
3.5 o
n
3.0
1.0 8 1.0 110 5.9 .0001 10 10.6 .00004 13 (2.2-15.8)* (3.1-36.3)* 14 4
.7
1.0 1.2 (0.3-4.2)*
99 24
.8
2.5 o ,~ c~. <
Abbreviations: AD, Alzheimer's disease; Poss, possible Alzheimer's disease; N, normal control; OR, odds ratio. *Parentheses indicate a 95% confidence interval.
2.0
AI3 D e p o s i t i o n in R e l a t i o n t o A p o E Polymorphism
I n Ad, cases with at least 1 c o p y o f e4 h a d signific a n d y i n c r e a s e d d e p o s i t s o f A[3 in n e o c o r t e x ( M a n n 4.0 3.5 3.0
o
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Figure 2. AI3 deposits in brain regions of Ad and Poss. Error bars show mean + SD. Size of circles represents number of cases with the same % area of deposits (see legend).
W h i t n e y , P = .014) (Fig 3). B e c a u s e t h e r e was o n l y 1 e4 h o m o z y g o t e , f u r t h e r c o m p a r i s o n o f t h e n u m b e r o f e4 alleles in r e l a t i o n to A[3 l o a d c o u l d n o t b e d o n e . Interestingly, t h e A ~ l o a d in this case was a b o v e t h e a v e r a g e o f A[3 d e p o s i t i o n in cases with 1 e4 allele. However, e4 was n o t a s s o c i a t e d with i n c r e a s e d A[3 l o a d in h i p p o c a m p u s ( M a n n - W h i t n e y , P = .50) o r a m y g d a l a ( P = .32). Cases with at least 1 c o p y o f e2 w e r e n o t a s s o c i a t e d with d e c r e a s e d A ~ d e p o s i t i o n in t h e b r a i n ( M a n n - W h i t n e y , P = .60 in h i p p o c a m p u s ; P = .15 in a m y g d a l a ; P = .15 in n e o c o r t e x ) . I n Poss, n e i t h e r e4 n o r E2 was a s s o c i a t e d with A[3
3.0 o
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0.0 Among neocortex, hippocampus, and amygdala, there w e r e s i g n i f i c a n t d i f f e r e n c e s in A[3 l o a d ( F r i e d m a n A N O V A , P < .05 f o r A d a n d P < .001 f o r Poss) (Fig 2), w h i c h w e r e d u e to t h e low level o f A[3 d e p o s i t i o n in t h e a m y g d a l a in A d ( W i ! c o x o n , a m y g d a l a v h i p p o c a m p u s , P = .007; a m y g d a ! a V n e o c o r t e x , P = .003; h i p p o c a m p u s v n e o c o r t e x , P = .82), a n d also in Poss ( a m y g d a l a v h i p p o c a m p u s , P = .013; a m y g d a l a v n e o c o r t e x , P = .012; h i p p o c a m p u s v n e o c o r t e x , P = .68). T h e r e was also d e c r e a s e d A[3 d e p o s i t i o n in e i t h e r h i p p o c a m p u s ( M a n n W h i t n e y , P = .041), a m y g d a l a ( P = .018), o r n e o c o r t e x ( P = .044) in t h e Poss g r o u p w h e n c o m p a r e d with A d (Fig. 2).
o
o
~"
1.0 0.7 (0.2-2.1) ~
o
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0
1
Number of ApoE alleles
Figure 1. AI3 deposits in neocortical regions of Ad and Poss. Error bars show mean _+standard deviation (SD). Size of circles represents number of cases with the same % area of deposits (see legend). 1174
Figure 3. A!B deposits in neocortex for Ad cases with or without ApoE ~4 alleles. Black dot denotes in ApoE e4 homozygore. Error bars show mean + SD.
LATE-ONSET ALZHEIMER'S DISEASE ( C h e n et al)
deposition in h i p p o c a m p u s (Mann-Whitney, P = .15 for e4 and P = .50 for e2), amygdala (P = .97 for e4 and P = .50 for e2) or n e o c o r t e x (P = .15 for e4 a n d P = .57 for ~2).
NFT Formation in AD Brains Neurofibrillary tangles (NFTs) are r e g a r d e d as a reference c o m p o n e n t for diagnosis by CERAD criteria. ~ In b o t h AD and Poss, tan staining m o s t frequently a p p e a r e d in h i p p o c a m p u s , less frequently in amygdala, and least frequently in the 4 regions of n e o c o r t e x (Fig 4). Because the quantity of NFTs was rather low in the neocortex, only data on tan immunoreactivity in h i p p o c a m p u s a n d amygdala were f u r t h e r analyzed. A m o n g the Ad cases, the density of NFTs in the h i p p o c a m p u s was greater t h a n in the amygdala (Wilcoxon, P = .005), whereas there was no significant difference between h i p p o c a m p u s a n d amygdala in the Poss cases (P = .20) (Fig 4). T h o u g h we f o u n d a trend toward m o r e NFTs in Ad than Poss, the difference was not significant in h i p p o c a m p u s (Mann-Whimey, P = .11) or amygdala ( P = .33). In b o t h Ad and Poss, tan staining was not correlated with A[3 load in h i p p o c a m pus ( S p e a r m a n correlation, R = 0.14, P = .44 for Ad; R = 0.26, P = 0.29 for Poss) or amygdala ( R - - - . 0 7 , P = .70 for Ad; R = 0.17, P = .49 for Poss).
o o 0
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.40
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1 case 2-3 cases 4-7 cases 8-15 cases >15 cases
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<~ Figure 4. N u m b e r of NFT per m m 2 in different brain regions of A d a n d Poss. Error bars show m e a n _+ (or m e a n + SD w h e n m e a n - SD is less t h a n 0), Size of circles represents n u m b e r of cases with t h e s a m e NFT density (see legend).
NFT Formation in Relation to ApoE Polymorphism T h e n u m b e r s of NFTs were not associated with ~4 in either Ad (Mann-Whitney, P = .82 in h i p p o c a m p u s , P -- .15 in amygdala) or Poss (P -- .57 in h i p p o c a m p u s , P = .57 in amygdala). Neither was tan deposition associated with e2 in either Ad (P = .85 in h i p p o c a m p u s , P = .23 in amygdala) or Poss (P = .38 in h i p p o c a m p u s , P = .44 in amygdala).
DISCUSSION In summary, we f o u n d ApoE e4 was a high risk factor in Chinese late-onset AD. e4 was associated with increased A[3 deposition in n e o c o r t e x in pathologically c o n f i r m e d definite or p r o b a b l e AD. NFTs were not associated with any ApoE genotype. Since e2 was first r e p o r t e d to be u n d e r r e p r e s e n t e d in whites, 2s its protective effect has r e m a i n e d controversial in o t h e r ethnic groups, including African Americans a n d Hispanics, 22 Japanese, 22 Dutch, 24 and Italians. 25 Similarly, we c a n n o t d e m o n s t r a t e a protective role of e2 in Chinese definite or p r o b a b l e AD. This result is compatible with o u r pathological observation that, in definite or p r o b a b l e AD, e2 allele is not associated with decreased A[3 plaque deposits in either neocortex, amygdala, or h i p p o c a m p u s . We have c o n f i r m e d ApoE E4 as a risk factor in Chinese definite or p r o b a b l e AD by frequency and odds ratio analyses, substantiating a previous r e p o r t on clinical samples in Chinese. 26 O u r odds ratio is similar to that calculated f r o m meta-analyses of o t h e r ethnic groups 22 (OR [CI] for Japanese, 4.3 [3.3-5.5]; Caucasians, 4.1 [3.8-4.4]; Hispanics, 2.2 [1.5-3.3]; and blacks, 2.0 [1.3-2.9]). ApoE e4 allele frequency in o u r controls is 5.3%, which is consistent with Mak et al.'s study 26 (7%) and a n o t h e r study on ApoE polymorphisms in relation to cholesterol level in different ethnic groups, including Chinese 27 (7%). In whites, however, the e4 frequency is as high a s 8 % - 2 1 % 6,11,22 in controls. Because ApoE e4 is the strongest genetic risk factor for AD, accounting for m o r e than 50% of all whites' late-onset sporadic AD cases, 2s the low frequency of E4 in Chinese might explain their low AD prevalence r e p o r t e d in general. 13,14 T h o u g h a descending o r d e r of frequency of NFTs f r o m h i p p o c a m p u s to amygdala and to the neocorrex similar to o t h e r studies 19 is f o u n d in o u r series, the overall n u m b e r s in the n e o c o r t e x remain small. This confirms a previous r e p o r t that NFTs are not very n u m e r o u s a m o n g AD brains in Chinese. 99 In o u r definite a n d p r o b a b l e AD, cases with e4 are associated with significantly increased A[3 plaque deposits in neocortex. T h e same trend occurs but is not significant with h i p p o c a m p u s or amygdala in our study. O u r study also shows a consistent pattern of A[~ plaque deposits evenly distributed t h r o u g h o u t 3 neocortical regions in definite or p r o b a b l e AD, a n d plaques are m o r e n u m e r o u s in n e o c o r t e x and h i p p o c a m p u s than in anaygdala. Widespread lesions in n e o c o r t e x may explain that in late-stage AD patients, language problems,
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HUMAN PATHOLOGY
Volume 30, No. 10 (October 1999)
visuospatial disorientation, and loss of insight and reasoning often develop. 3° Similar studies on whites also showed a roughly h o m o g e n e o u s pattern of A[3 deposition t h r o u g h o u t the neocortex. 31 Plaque deposits have been shown to be significantly associated with ApoE e4 in middle frontal cortex and hippocampus (CA1) in Schmechel et al's study, 7 in superior temporal sulcus in the Gomez-Isla et al t~ report, 9 in the average of middle frontal, superior temporal, middle temporal, and inferior parietal cortex in Polvikoski et al's data, 6 only in n e o c o r t e x (average of frontal and parietal cortex), but not in hippocampus (CA1) or entorhinal cortex in Lippa et al's paper. ~2 Interestingly, a large recent autopsy series by Berg et al s failed to find a relationship between ~4 and plaque load at neocortex. However, some studies did not use A[3 immunostaining, which might account for the discrepancies, s,l°-12 T h a t ApoE e4 genotype is associated with increased A[3 deposition in n e o c o r t e x corresponds to the in vitro observations that ApoE4 binds to A[3 more avidly than ApoE3 sa and that ApoE4 has a strong stimulatory effect on the polymerization of A[3 into amyloid filaments. 34 In our study, NFTs as demonstrated by tau immunostaining are not associated with ApoE e4 in pathologically diagnosed definite or probable AD. It has b e e n hypothesized that ApoE3 might interact with tau by preventing it from h y p e r p h o s p h o r y l a t i o n , whereas ApoE4 does not. 5 However, results from studies on ApoE genotype in relation to NFT load in AD patients are controversial. Schmechel et al 7 and Polvikoski et al 6 observed increased NFT in cases with the e4 allele, whereas Gomez-Isla et al 9 did not, and neither did we. Thus, the involvement of ApoE4 in NFT formation requires further studies. NFTs are not associated with A[3 load in our definite and probable AD. This suggests that NFTs and amyloid plaques, the 2 major pathological lesions in AD pathology, might play roles in neurodegeneration through i n d e p e n d e n t mechanisms. In this study, cases of possible AD share the same pattern of ApoE alleles distribution and follow the same pattern of A[3 and tau deposition in brain regions as in definite or probable AD, although the overall A[3 load is increased in the latter. T h e lower A[3 level in the possible AD group might reflect a delay between the appearance of pathology and the onset of dementia. This lends support to the hypothesis that Alzheimer's lesions in many instances might represent preclinical AD. Had patients lived longer, more A[3 plaques aggregate, and clinical manifestation of dementia might develop. Interestingly, the m e a n age at death of possible AD is almost 4 years younger than that of definite or probable AD in our study (Mann-Whitney, P = .18). A separate prospective study also suggests that changes in possible AD may represent pathological aging and early AD, because n o n d e m e n t e d elderly classified as possible AD after autopsy show impaired secondary m e m o r y and frontal executive function, which are measures sensitive to early-stage A D Y These findings support the view of the recent Consensus Report that any Alzheimer-type pathological changes are pathological even when they appear to be incidental. 4 The relatively low levels of
pathological lesions in possible cases might hide a relation between A[3 deposition and ApoE genotype in our study. The results from these possible cases are pending corroboration from other groups.
REFERENCES 1. Saunders AM, Hulette O, Weish-Bohmer KA, et al: Specificity, sensitivity, and predictive value of apolipoprotein-E genotyping for sporadic Alzheimer's disease. Lancet 348:90-93, 1996 2. Mirra SS, Heyman A, McKeel D, et al: The consortium to establish a registroy for Alzheimer's disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease. Neurology 41:479-486, 1991 3. Mirra SS, Hart MN, Terry RD: Making the diagnosis of Alzheimer's disease. Arch Pathol Lab Med 117:132-144, 1993 4. Hyman BT, TrojanowskiJQ: Editorial on consensus recommendations for the postmortem diagnosis of Alzheimer disease from the National Institute on Aging and the Reagan Institute Working Group on diagnostic criteria for the neuropathological assessment of Alzheimer disease.J Neuropathol Exp Neurol 56:1095-1097, 1997 5. Wasco W, Tanzi RE: Molecular mechanisms of dementia. Totowa, New Jersey, Humana Press, 1997, pp 21-37 6. Polvikoski T, Sulkava R, Haltia M, et al: Apolipoprotein E, dementia, and cortical deposition of [3-amyloid protein. N EnglJ Med 333:1242-1247, 1995 7. Schmechel DE, Saunders AM, Strittmatter WJ, et al: Increased amyloid [3-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease. Proc Natl Acad Sci U S A 90:9649-9653, 1993 8. Berg L, McKeel DW, MillerJP, et al: Clinicopathologic studies in cognitively healthy aging and Alzheimer disease. Arch Neurol 55:326-335, 1998 9. Gomez-Isla T, West HL, Rebeck GW, et al: Clinical and pathological correlates of apolipoprotein E e4 in Alzheimer's disease. Ann Neurol 39:62-70, 1996 1O. Itoh Y, Yamada M, Polvikoski T, et al: Apolipoprotein E and the neuropathology of dementia. N EnglJ Med 334:599-600, 1996 11. Landen M, Thorsell A, Wallin A, et al: The apolipoprotein E allele epsilon 4 does not correlate with the n u m b e r of senile plaques or neurofibrillary tangles in patients with Alzheimer's disease. J Neurol Neurosurg Psychiatry 61:352-356, 1996 12. Pirttil/i T, Soininen H, Mehta PD, et al: Apolipoprotein E genotype and amyloid load in Alzheimer disease and control brains. Neurobiology of Aging 18:121-127, 1997 13. Chiu HFK, Lam LCW, Chi I, et al: Prevalence of dementia in Chinese elderly in Hong Kong. Neurology 50:1002-1009, 1997 14. Zhang MY, Katzman R, Salmon D, et al: The prevalence of dementia and Alzheimer's disease in Shanghai, China: Impact of age, gender and education. Ann Neurol 27:428-437, 1990 15. Evans DA, Funkenstein HH, Albert MS, et al: Prevalence of Alzheimer's disease in a community population of older persons: Higher than previously reported (see comments). JAMA 262:25512556, 1989 16. Rocca WA, Hofman A, Brayne C, et al: Frequency and distribution of Alzheimer's disease in Europe: A collaborative study of 1980-1990 prevalence findings: The EURODEM-Prevalence Research Group. Ann Neurol 30:381-390, 1991 17. Ng HK: Alzheimer's disease and Alzheimer-type of cerebral degenerations in Chinese. Clin Exp Neurol 31:85-98, 1994 18. Ng HK, Lee JCK: Degenerative cerebral alterations in Chinese aged 65 years or older. Clin Neuropathol 7:280-284, 1988 19. Braak H, Braak E: Neuropathological stageing of Alzheimerrelated changes. Acta Neuropathol 82:239-259, 1991 20. Sambrook J, Eritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, (ed 2). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1989, pp E3-E4, E10-Ell, B.16 21. Kontula K, Aalto-Setala K, Kuusi T, et al: Apolipoprotein E polymorphism determined by restriction enzyme analysis of DNA amplified by polymerase chain reaction: Convenient alternative to phenotyping by isoelectric focusing. Clin Chem 36:2087-2092, 1990
1176
LATE-ONSET ALZHEIMER'S DISEASE(Chen et al) 22. Farrer LA, Cupples LA, Haines JL, et al: Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease: A meta-analysis. JAMA 278:1349-1356, 1997 23. Corder EH, Saunders AM, Risch NJ, et al: Protective effect of apolipoprotein E type 2 allele for late onset Alzheimer disease. Nature Genet 7:180-184, 1994 24. Van Duijin C, de Knijff E Wehnert A, et al: The apolipoprorein E epsilon 2 allele is associated with an increased risk of early-onset Alzheimer's disease and a reduced survival. Ann Neurol 37:605-610, 1995 25. Sorbi S, Nacmias B, Forleo P, et al: ApoE allele frequencies in Italian sporadic and familial Alzheimer's disease. Neurosci Lett 177:100-102, 1994 26. MakYT, Chiu H, Woo J, et al: Apolipoprotein E genotype and Alzheimer's disease in Hong Kong elderly Chinese. Neurology 46:146149, 1996 27. Hallman DM, Boerwinkle E, Saha N, et ah The apolipoprotein E polymorphism: A comparison of allele frequencies and effects in nine populations. A m J Hum Genet 49:338-349, 1991 28. Nalbantoglu J, Gilfix B, Bertrand P, et al: Predictive value of apolipoprotein E genotyping in Alzheimer's disease: Results of an autopsy series and an analysis of several combined studies. Ann Neurol 36:889-895, 1994
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29. Ng HK, Cheng Y, Poon WS: Alzheimer-type of pathological changes in Chinese.J Neurol Sci 145:97-103, 1997 30. Heimer L: The Human Brain and Spinal Cord: Functional Neuroanatomy and Dissection Guide (ed 2). NewYork, NY, SpringerVerlag, 1998, pp 415-431 31. Arnold SE, Hyrnan BT, FloryJ, et al: The topographical and neuroanatomical distribution of neurofibrillary tangles and neuritic plaques in the cerebral cortex of patients with Alzheimer's disease. Cerebral Cortex 1:103-116, 1991 32. Lippa CR, Smith TW, Sauders AM, et al: Apolipoprotein E-~2 and Alzheimer's disease: Genotype influences pathologic phenotype. Neurology 48:515-519, 1997 33. Strittmatter WJ, Weisgraber KH, Huang DY, et al: Binding of h u m a n apolipoprotein E to synthetic amyloid [3 peptide: Isoformspecific effects and implications for late-onset Alzheimer disease. Proc Nail Acad Sci U S A 90:8098-8102, 1993 34. Sanan DA, Weigraber KH, Russell SJ, et ah Apolipoprotein E associates with beta amyloid peptide of Alzheimer's disease to form novel monofibrils: Isoform ApoE 4 associates more efficiently than ApoE 3.J Clin Invest 94:860-869, 1994 35. Hulette CM, Welsh-Bohmer KA, Murray MG, et al: Neuropathological and neuropsychological changes in "normal" aging: Evidence for preclinical Alzheimer disease in cognitively normal individuals. J Neuropathol Exp Neurol 57:1168-1174, 1998
significantly increased in the neocortex in Ad examined with at least 1 copy of ~4 compare d with subjects without ~4 (Mann-Whitney, P = .014). The same trend, though not statistically significant, occurred in Poss (P = .15). Tan expression was associated with ApoE ~4 in neither Ad nor Poss. Poss is genetically and histologically similar to Ad, although the overall A[~ load is significantly increased in the latter. These findings support the recent Consensus Report's findings that all Alzheimer-type pathology may be significant. HUM PATUOL30:11721177. Copyright © 1999 byW.B. Saunders Company Key words:Alzheimer's disease, 13-amyloid plaque, neurofibriliary tangle, apolipoprotein E, possible AD. Abbreviations: AD, Alzheimer's disease; CERAD, the Consortium to Establish a Registry for Alzheimer's Disease; ApoE, apolipoprotein E; A13, 13 amyloid; NFI', neurofibrillary tangle; Ad, definite or probable Alzheimer's disease; Poss, possible Alzheimer's disease; N, normal controls; PAP, peroxidase anti-peroxidase; PCR, polymerase chain reaction; OR, odds ratio; CI, 95% confidence intervals.
A diagnosis of definite Alzheimer's disease (AD) requires confirmation by histology and autopsy even though the clinical diagnosis for AD generally exceeds 80% accuracy) Although histological criteria remain imperfect, neuropathologists involved in the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) established simple and practical neuropathological criteria for AD in 1991 z and updated them in 19933 and 1997. 4 Criteria for levels of AD diagnosis from definite, probable, to possible AD are provided, and they d e p e n d on the presence or absence of a history of dementia and the severity of neuritic plaques in relation to age. In a general hospital where not all elderly patients are assessed carefully as to their mental status and subtle mental deterioration may be overlooked, autopsy brains that demonstrate significant Alzheimertype pathology may be categorized as possible AD (Poss). Currently, there have been few studies on these possible AD cases. Although the apolipoprotein E (ApoE) e4 allele is known to be a strong risk factor for AD, how its presence affects the development of AD is still uncertain. ApoE
has a high avidity to [3 amyloid (A~), and it is found in neuritic plaques and some neurons. It is hypothesized that ApoE4 might be involved in AD pathogenesis by its effects on amyloid aggregation in neuritic plaques or formation of paired helical filaments in the neurofibrillary tangle (NFT). 5 A[3 plaque deposits and NFTs have been observed to be increased in brains of AD patients with ~4 compared with patients without e4. 6,7 However, the association of ApoE e4 with AD pathology is still controversial according to other authors 8-12and deserves further study. Studies suggest that there might be a difference in AD between Chinese and Western populations. AD accounts for approximately 4% of the population older than age 70 years in H o n g Kong is and 3% older than 65 years in Shanghai. 14 These figures are at the lower margin of the prevalence reported in Americans ]~ (10 % over the age of 65) and Europeans TM (3% to 11% over the age of 60). Alzheimer-type change in brains from n o n d e m e n t e d elderly has also been found to be less than that in Caucasians. 17,1s However, there have been no studies correlating different ApoE genotypes with postmortem AD changes in Chinese. In this study, we attempted to perform ApoE genotyping and examine its relationship to A[3 plaque deposits and NFT formation in different areas of autopsy brains in Chinese late onset AD. We also tried to delineate the ApoE genetics and the pathological characteristics of Poss, which to our knowledge has not previously been performed in Chinese.
From the Department of Anatomical and Cellular Pathology, Department of Ophthalmology and Visual Sciences, Department of Chemical Pathology, Chinese University of Hong Kong, Shatin, Hong Kong. Accepted for publication May 12, 1999. Address correspondence and reprint requests to Ho-keung Ng, MD, FRCPath, FRCPA, Department of Anatomical & Cellular Pathology, Prince of Wales Hospital, Shatin, Hong Kong. Copyright © 1999 by W.B. Saunders Company 0046-8177/99/3010-0009510.00/0
1172
LATE-ONSETALZHEIMER'SDISEASE(Chen et al) MATERIALS AND METHODS
Subjects Thirty-four autopsy brains of definite or probable AD (Ad) with mean age of 82.8 + 7.9 years (standard deviation), 18 cases of Poss having no clinical history of dementia with mean age 78.7 -+ 9.6, and 123 normal controls (N) with mean age 82.0 +- 6.6 were collected in our hospital during 1992 through 1997. Diagnosis was previously made based on CERAD criteria by Bielschowsky and Bodian staining of sections from hippocampus (including entorhinal cortex), amygdala, superio r temporal lobe, middle frontal gyrus, and inferior parietal lobule, s and medial occipital lobe according to Braak and Braak} 9
AI~ and Tau Immunostaining of AD Patients Paraffin sections were cut at a thickness of 4 pm for immunostaining. In each AD case, sections from 1 hippocampus (including entorhinal cortex), amygdala, superior temporal gyrus, middle frontal gyrus, and inferior parietal lobule were stained with a monoclonal antibody against A[3 (DAKO, 1/50) by the avidin-biotin complex method and counterstained by methyl green. The above regions plus sections from 1 medial occipital lobe were stained with a polyclonal antibody against tau for NFTs (DAKO, 1/600, with overnight incubation) by the peroxidase anti-peroxidase (PAP) method. Cases of definite AD were used as positive controls. For negative control, the primary antibody was replaced by 5% normal rabbit serum in the ABC m e t h o d and 2% normal swine serum in the PAP method.
72°C for 5 minutes. One microliter of a 50-fold-dilution of the first PCR product was used for a second PCR by P2 and P3 (Gibco-BRL) in a final volmne of 30 pL with the conditions described, except that 35 cycles of 30 seconds at 95°C, 1 minute at 68°C, and 1 minute at 72°C were used. An aliquot of 25 pL of the second PCR products was digested by 2 U of Hha I (Gibco-BRL) overnight, followed by electrophoresis on 12% polyacrylamide gels as described21 The fragments were visualized by ultraviolet light after staining with ethidium bromide. All cases were successfully genotyped.
StatisticalCalculations For ApoE genotyping, chi-squares, odds ratios (OR), and 95% confidence intervals (CI) were calculated using the Epi6 computer program (Centers for Disease Control, Atlanta, GA). Yates-corrected P values were used unless the expected values were less than 5, when Fisher's exact p values were used instead. Nonparametric tests were used for pathological data analysis using Statistica software (Statsoft, Tulsa, OK).
RESULTS ApoE Polymorphism
A[~ plaque deposits were determined by calculating the percentage area covered by A[Mmmunostained plaques at a magnification of ×100 by using the GAS 200-cell analysis system (Becton Dickinson, Franklin Lakes, NJ). In each of the 5 brain regions mentioned above, 15 to 20 microscopic fields with the most numerous A[3 plaques were counted, and the percentage area occupied by A[3 staining was averaged.
A p o E e4 was significantly i n c r e a s e d in A d o r Poss versus N (X2, d f = 1, P = .00005 f o r Ad; P = .000005 f o r Poss), with a n o d d s r a t i o f o r cases with at least 1 c o p y o f t h e e4 allele c o m p a r e d with cases w i t h o u t ~4 o f 5.9 times f o r AD a n d 10.6 t i m e s f o r Poss (Table 1, 2). As t h e least c o m m o n allele in C h i n e s e , e4 was f o u n d in 5.3% o f c o n t r o l s in this study (Table 2). e2, t h e least c o m m o n allele in whites, 22 was m o r e c o m m o n t h a n ~4 in N in this study. However, it was n o t u n d e r r e p r e s e n t e d in A d o r Poss cases as c o m p a r e d with N by X2 ( d f = 1, P = .77 for Ad, P = 1.00 f o r Poss) a n d o d d s r a t i o analysis (Table 2). A d a n d Poss d i d n o t significantly differ f r o m e a c h o t h e r in t h e d i s t r i b u t i o n o f t h e 3 A p o E alleles (X2, d f = 2, P = .38).
Counting of NFTs
A!3 Deposition in AD Brains
NFTs were counted with an eyepiece graticule at 100× magnification of a Nikon Labophot 2 microscope. In each of the 6 brain regions stained for tau, 15 to 20 microscopic fields of 1 mm 2 containing the most numerous NFTs were counted and an average obtained as NFT density.
I n b o t h A d a n d Poss, A[3 d e p o s i t i o n q u a n t i f i e d as p e r c e n t a g e a r e a was s t u d i e d in 3 r e g i o n s o f n e o c o r t e x , h i p p o c a m p u s , a n d a m y g d a l a f o r e a c h case. T h e r e was n o s i g n i f i c a n t d i f f e r e n c e o f A[3 l o a d a m o n g t h e f r o n t a l , t e m p o r a l , a n d p a r i e t a l cortices in A d o r Poss ( F r i e d m a n ANOVA, P < .54 for A d a n d P < .18 f o r Poss), i n d i c a t i n g t h a t A[3 d e p o s i t s were evenly d i s t r i b u t e d in t h e 3 r e g i o n s o f n e o c o r t e x (Fig 1). T h e r e f o r e , in e a c h case, A[3 l o a d s in t h e 3 r e g i o n s w e r e a v e r a g e d as " n e o c o r t e x " to c o m p a r e with t h a t in h i p p o c a m p u s a n d a m y g d a l a .
Quantification of AI3 Plaque Deposits
ApoE Genotyping For each case, DNA was extracted from 5 pieces of 10-1Jm paraffin tissue sections by digestion with proteinase K (BULK, 10 m g / m L , overnight at 37°C), p h e n o l / c h l o r o f o r m treatment, and precipitation with cold ethanol as described, z° The fourth exon of the ApoE gene was amplified by the two-step nested polymerase chain reaction (PCR) using primers as previously described. 21 The first PCR by primers P1 and P4 (Gibco-BRL), contains about 500 ng genomic DNA, 200 p m o l / L of each dNTE 0.5 lamol/L of each primer, 3.0 m m o l / L magnesium chloride, 10% dimethylsulfoxide, 0.004% gelatin, and 1 U Taq DNA polymerase in a final volume of 20 IlL. After initial denaturing at 95°C for 5 minutes, a touchdown PCR program was carried out for 40 cycles of 30 seconds at 95°C, 30 seconds at 72°C, decreasing 0.4°C per cycle and 1 minutes at 72°C, followed by a final extension at
TABLE 1. ApoEAllele and Genotype Distribution Alleles e3 (%)
e2 (%)
Oenotype e4 (%)
AD 47 (69.1) 6 (8.8) 15 (22.1) Poss 20 (55.6) 4 (11.1) 12 (33.3) N 206 (83.7) 27 (11.0) 13 (5.3)
2/2 2/3 3/3 2/4 3/4 4/4 1 0 3
4 3 21
15 5 86
0 1 0
13 7 13
1 2 0
Abbreviations: AD, Mzheimer's disease; Poss, possible Alzheimer's disease; N, normal controls.
1173
HUMAN PATHOLOGY
Volume 30, No. 10 (October 1999)
TABLE 2.
Odds Ratios for Alzheimer's Disease in Association With G e n o t y p e s With or Without ApoE e4 Alleles
AD n
Poss
OR
Copies of e4 0 20 1 or 2 14 Copies of e2 0 29 1 or 2 5
p
n
4.5 4.0 N
OR
p
3.5 o
n
3.0
1.0 8 1.0 110 5.9 .0001 10 10.6 .00004 13 (2.2-15.8)* (3.1-36.3)* 14 4
.7
1.0 1.2 (0.3-4.2)*
99 24
.8
2.5 o ,~ c~. <
Abbreviations: AD, Alzheimer's disease; Poss, possible Alzheimer's disease; N, normal control; OR, odds ratio. *Parentheses indicate a 95% confidence interval.
2.0
AI3 D e p o s i t i o n in R e l a t i o n t o A p o E Polymorphism
I n Ad, cases with at least 1 c o p y o f e4 h a d signific a n d y i n c r e a s e d d e p o s i t s o f A[3 in n e o c o r t e x ( M a n n 4.0 3.5 3.0
o
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o
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2.0
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< 1.0 0.5
o
o
o
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Amygdala
Figure 2. AI3 deposits in brain regions of Ad and Poss. Error bars show mean + SD. Size of circles represents number of cases with the same % area of deposits (see legend).
W h i t n e y , P = .014) (Fig 3). B e c a u s e t h e r e was o n l y 1 e4 h o m o z y g o t e , f u r t h e r c o m p a r i s o n o f t h e n u m b e r o f e4 alleles in r e l a t i o n to A[3 l o a d c o u l d n o t b e d o n e . Interestingly, t h e A ~ l o a d in this case was a b o v e t h e a v e r a g e o f A[3 d e p o s i t i o n in cases with 1 e4 allele. However, e4 was n o t a s s o c i a t e d with i n c r e a s e d A[3 l o a d in h i p p o c a m p u s ( M a n n - W h i t n e y , P = .50) o r a m y g d a l a ( P = .32). Cases with at least 1 c o p y o f e2 w e r e n o t a s s o c i a t e d with d e c r e a s e d A ~ d e p o s i t i o n in t h e b r a i n ( M a n n - W h i t n e y , P = .60 in h i p p o c a m p u s ; P = .15 in a m y g d a l a ; P = .15 in n e o c o r t e x ) . I n Poss, n e i t h e r e4 n o r E2 was a s s o c i a t e d with A[3
3.0 o
tO iOi Ii
2.5
o
O
I
2.0
o
ii ° oo
o o
o
o
o
Ad Poss Frontal
o
o o
Ad Poss Parietal
o Ca.
o O
o
~'
o
o
1.5
e~
<
T
o
0.0
o
o
Hippocampus
Neocortex
o
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o
o
o
o
rt~
to
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o o
o o
O it ! !i I o
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1.5
0.0 Among neocortex, hippocampus, and amygdala, there w e r e s i g n i f i c a n t d i f f e r e n c e s in A[3 l o a d ( F r i e d m a n A N O V A , P < .05 f o r A d a n d P < .001 f o r Poss) (Fig 2), w h i c h w e r e d u e to t h e low level o f A[3 d e p o s i t i o n in t h e a m y g d a l a in A d ( W i ! c o x o n , a m y g d a l a v h i p p o c a m p u s , P = .007; a m y g d a ! a V n e o c o r t e x , P = .003; h i p p o c a m p u s v n e o c o r t e x , P = .82), a n d also in Poss ( a m y g d a l a v h i p p o c a m p u s , P = .013; a m y g d a l a v n e o c o r t e x , P = .012; h i p p o c a m p u s v n e o c o r t e x , P = .68). T h e r e was also d e c r e a s e d A[3 d e p o s i t i o n in e i t h e r h i p p o c a m p u s ( M a n n W h i t n e y , P = .041), a m y g d a l a ( P = .018), o r n e o c o r t e x ( P = .044) in t h e Poss g r o u p w h e n c o m p a r e d with A d (Fig. 2).
o
o
~"
1.0 0.7 (0.2-2.1) ~
o
1.0
o
o
0.5 0.0
Ad P o s s Temporal
0
1
Number of ApoE alleles
Figure 1. AI3 deposits in neocortical regions of Ad and Poss. Error bars show mean _+standard deviation (SD). Size of circles represents number of cases with the same % area of deposits (see legend). 1174
Figure 3. A!B deposits in neocortex for Ad cases with or without ApoE ~4 alleles. Black dot denotes in ApoE e4 homozygore. Error bars show mean + SD.
LATE-ONSET ALZHEIMER'S DISEASE ( C h e n et al)
deposition in h i p p o c a m p u s (Mann-Whitney, P = .15 for e4 and P = .50 for e2), amygdala (P = .97 for e4 and P = .50 for e2) or n e o c o r t e x (P = .15 for e4 a n d P = .57 for ~2).
NFT Formation in AD Brains Neurofibrillary tangles (NFTs) are r e g a r d e d as a reference c o m p o n e n t for diagnosis by CERAD criteria. ~ In b o t h AD and Poss, tan staining m o s t frequently a p p e a r e d in h i p p o c a m p u s , less frequently in amygdala, and least frequently in the 4 regions of n e o c o r t e x (Fig 4). Because the quantity of NFTs was rather low in the neocortex, only data on tan immunoreactivity in h i p p o c a m p u s a n d amygdala were f u r t h e r analyzed. A m o n g the Ad cases, the density of NFTs in the h i p p o c a m p u s was greater t h a n in the amygdala (Wilcoxon, P = .005), whereas there was no significant difference between h i p p o c a m p u s a n d amygdala in the Poss cases (P = .20) (Fig 4). T h o u g h we f o u n d a trend toward m o r e NFTs in Ad than Poss, the difference was not significant in h i p p o c a m p u s (Mann-Whimey, P = .11) or amygdala ( P = .33). In b o t h Ad and Poss, tan staining was not correlated with A[3 load in h i p p o c a m pus ( S p e a r m a n correlation, R = 0.14, P = .44 for Ad; R = 0.26, P = 0.29 for Poss) or amygdala ( R - - - . 0 7 , P = .70 for Ad; R = 0.17, P = .49 for Poss).
o o 0
50
~E E
.40
o
1 case 2-3 cases 4-7 cases 8-15 cases >15 cases
o
o o
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20 Z
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o 10
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o~
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~
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o
o
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~.~
o
<~ Figure 4. N u m b e r of NFT per m m 2 in different brain regions of A d a n d Poss. Error bars show m e a n _+ (or m e a n + SD w h e n m e a n - SD is less t h a n 0), Size of circles represents n u m b e r of cases with t h e s a m e NFT density (see legend).
NFT Formation in Relation to ApoE Polymorphism T h e n u m b e r s of NFTs were not associated with ~4 in either Ad (Mann-Whitney, P = .82 in h i p p o c a m p u s , P -- .15 in amygdala) or Poss (P -- .57 in h i p p o c a m p u s , P = .57 in amygdala). Neither was tan deposition associated with e2 in either Ad (P = .85 in h i p p o c a m p u s , P = .23 in amygdala) or Poss (P = .38 in h i p p o c a m p u s , P = .44 in amygdala).
DISCUSSION In summary, we f o u n d ApoE e4 was a high risk factor in Chinese late-onset AD. e4 was associated with increased A[3 deposition in n e o c o r t e x in pathologically c o n f i r m e d definite or p r o b a b l e AD. NFTs were not associated with any ApoE genotype. Since e2 was first r e p o r t e d to be u n d e r r e p r e s e n t e d in whites, 2s its protective effect has r e m a i n e d controversial in o t h e r ethnic groups, including African Americans a n d Hispanics, 22 Japanese, 22 Dutch, 24 and Italians. 25 Similarly, we c a n n o t d e m o n s t r a t e a protective role of e2 in Chinese definite or p r o b a b l e AD. This result is compatible with o u r pathological observation that, in definite or p r o b a b l e AD, e2 allele is not associated with decreased A[3 plaque deposits in either neocortex, amygdala, or h i p p o c a m p u s . We have c o n f i r m e d ApoE E4 as a risk factor in Chinese definite or p r o b a b l e AD by frequency and odds ratio analyses, substantiating a previous r e p o r t on clinical samples in Chinese. 26 O u r odds ratio is similar to that calculated f r o m meta-analyses of o t h e r ethnic groups 22 (OR [CI] for Japanese, 4.3 [3.3-5.5]; Caucasians, 4.1 [3.8-4.4]; Hispanics, 2.2 [1.5-3.3]; and blacks, 2.0 [1.3-2.9]). ApoE e4 allele frequency in o u r controls is 5.3%, which is consistent with Mak et al.'s study 26 (7%) and a n o t h e r study on ApoE polymorphisms in relation to cholesterol level in different ethnic groups, including Chinese 27 (7%). In whites, however, the e4 frequency is as high a s 8 % - 2 1 % 6,11,22 in controls. Because ApoE e4 is the strongest genetic risk factor for AD, accounting for m o r e than 50% of all whites' late-onset sporadic AD cases, 2s the low frequency of E4 in Chinese might explain their low AD prevalence r e p o r t e d in general. 13,14 T h o u g h a descending o r d e r of frequency of NFTs f r o m h i p p o c a m p u s to amygdala and to the neocorrex similar to o t h e r studies 19 is f o u n d in o u r series, the overall n u m b e r s in the n e o c o r t e x remain small. This confirms a previous r e p o r t that NFTs are not very n u m e r o u s a m o n g AD brains in Chinese. 99 In o u r definite a n d p r o b a b l e AD, cases with e4 are associated with significantly increased A[3 plaque deposits in neocortex. T h e same trend occurs but is not significant with h i p p o c a m p u s or amygdala in our study. O u r study also shows a consistent pattern of A[~ plaque deposits evenly distributed t h r o u g h o u t 3 neocortical regions in definite or p r o b a b l e AD, a n d plaques are m o r e n u m e r o u s in n e o c o r t e x and h i p p o c a m p u s than in anaygdala. Widespread lesions in n e o c o r t e x may explain that in late-stage AD patients, language problems,
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Volume 30, No. 10 (October 1999)
visuospatial disorientation, and loss of insight and reasoning often develop. 3° Similar studies on whites also showed a roughly h o m o g e n e o u s pattern of A[3 deposition t h r o u g h o u t the neocortex. 31 Plaque deposits have been shown to be significantly associated with ApoE e4 in middle frontal cortex and hippocampus (CA1) in Schmechel et al's study, 7 in superior temporal sulcus in the Gomez-Isla et al t~ report, 9 in the average of middle frontal, superior temporal, middle temporal, and inferior parietal cortex in Polvikoski et al's data, 6 only in n e o c o r t e x (average of frontal and parietal cortex), but not in hippocampus (CA1) or entorhinal cortex in Lippa et al's paper. ~2 Interestingly, a large recent autopsy series by Berg et al s failed to find a relationship between ~4 and plaque load at neocortex. However, some studies did not use A[3 immunostaining, which might account for the discrepancies, s,l°-12 T h a t ApoE e4 genotype is associated with increased A[3 deposition in n e o c o r t e x corresponds to the in vitro observations that ApoE4 binds to A[3 more avidly than ApoE3 sa and that ApoE4 has a strong stimulatory effect on the polymerization of A[3 into amyloid filaments. 34 In our study, NFTs as demonstrated by tau immunostaining are not associated with ApoE e4 in pathologically diagnosed definite or probable AD. It has b e e n hypothesized that ApoE3 might interact with tau by preventing it from h y p e r p h o s p h o r y l a t i o n , whereas ApoE4 does not. 5 However, results from studies on ApoE genotype in relation to NFT load in AD patients are controversial. Schmechel et al 7 and Polvikoski et al 6 observed increased NFT in cases with the e4 allele, whereas Gomez-Isla et al 9 did not, and neither did we. Thus, the involvement of ApoE4 in NFT formation requires further studies. NFTs are not associated with A[3 load in our definite and probable AD. This suggests that NFTs and amyloid plaques, the 2 major pathological lesions in AD pathology, might play roles in neurodegeneration through i n d e p e n d e n t mechanisms. In this study, cases of possible AD share the same pattern of ApoE alleles distribution and follow the same pattern of A[3 and tau deposition in brain regions as in definite or probable AD, although the overall A[3 load is increased in the latter. T h e lower A[3 level in the possible AD group might reflect a delay between the appearance of pathology and the onset of dementia. This lends support to the hypothesis that Alzheimer's lesions in many instances might represent preclinical AD. Had patients lived longer, more A[3 plaques aggregate, and clinical manifestation of dementia might develop. Interestingly, the m e a n age at death of possible AD is almost 4 years younger than that of definite or probable AD in our study (Mann-Whitney, P = .18). A separate prospective study also suggests that changes in possible AD may represent pathological aging and early AD, because n o n d e m e n t e d elderly classified as possible AD after autopsy show impaired secondary m e m o r y and frontal executive function, which are measures sensitive to early-stage A D Y These findings support the view of the recent Consensus Report that any Alzheimer-type pathological changes are pathological even when they appear to be incidental. 4 The relatively low levels of
pathological lesions in possible cases might hide a relation between A[3 deposition and ApoE genotype in our study. The results from these possible cases are pending corroboration from other groups.
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