CA2111503A1 - Apolipoprotein e polymorphism and alzheimer's disease - Google Patents
Apolipoprotein e polymorphism and alzheimer's diseaseInfo
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- CA2111503A1 CA2111503A1 CA 2111503 CA2111503A CA2111503A1 CA 2111503 A1 CA2111503 A1 CA 2111503A1 CA 2111503 CA2111503 CA 2111503 CA 2111503 A CA2111503 A CA 2111503A CA 2111503 A1 CA2111503 A1 CA 2111503A1
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Abstract
The present invention relates to a method for the clinical determination of the risk for the late-onset of Alzheimer's disease of a patient, which comprises, determining the number of copies of the apo E gene allele E4 in a biological sample of said patient using an appropriate E4 probe, wherein zero, one or two copies of E4 indicates level of incidence of late-onset of Alzheimer's disease. The method of the present invention also provides for the prognosis for the treatment of Alzheimer's wherein one copy of E4 indicates a level of choline acetyltransferase reduced by half.
Description
- 2111S~3 APOLIPOPROTEIN E POLYMORPHISM AND ALZHEIMER'S DISEASE
BACKGROUND OF THE lNv~N~lION
Apolipoprotein E (apo E) is a well character-ized lipophilic protein associated with plasma and CSFlipoproteins. Apo E is synthesized primarily by the liver, but also at other sites including brain, macro-phages and adrenals (1). Furthermore, apo E is unique among apolipoproteins in that it is has a special rele-vance to the central and peripheral nervous systems.Apolipoprotein E (apoE) is important in modulating cholesterol and phospholipid transport from one cell to another. It is a key determinant in the cellular recognition and internalization of cholesterol-rich lipoproteins in the developing brain and in the response to neuronal injury (2,3,4,5). It also plays a fundamental role in the central nervous system (CNS) during synaptic remodelling induced by neuronal differentiation (2,3,6).
The apo E gene is located on chromosome 19, within a region which had previously been associated with familial late-onset cases of Alzheimer's disease (7). The structural gene for apo E is polymorphic, the three most common isoforms coded for are designated E2, E3 and E4 (5). These isoforms differ by amino acid substitutions at one or both of two sites, residues 112 and 158. The E2 isoform has Cys residues at sites 112 and 158. E3 has a Cys residue at site 112 and an Arg at site 158, and E4 has an Arg residue at sites 112 and 158. An individual thus may be homozygous (E2/2, E3/3 or E4/4) or heterozygous (E4/2, E4/3 or E3/2).
Apo E has been shown to be present in plaques and dystrophic neurites that characterize the neuropil of the Alzheimer's brain (8,9). Although apo E mRNA
and protein content is increased in response to neuro-~111503 nal cell loss in rat (2,3) and during demylination inhumans with multiple sclerosis (10), apo E expression remains relatively unchanged in the hippocampus of Alzheimer's (AD) patients (11) despite marked neuronal cell loss and differentiation in this structure (12).
Recent analysis of the apo E polymorphism in sporadic cases of Alzheimer's disease (13,14) has shown an increased frequency of the apo E4 allele in these indi-viduals. Moreover, there is a correlation between the age of onset for the disease and the number of copies of the E4 allele in that E4 homozygotes tend to have an earlier age of onset (13). Increased frequencies of the E4 allele have also been reported in familial cases of late onset AD (15).
Although the concept of subtypes of AD remains controversial to date,;it is clear that a significant portion of the AD population shows extrapyramidal symp-toms and neuropathological changes consistent with idiopathic Parkinson's disease (PD) (16, 17). The esti-mated prevalence of dementia in PD varies from 10% to 40% (18), the combination of AD/PD pathological fea-tures being common.
The present study was designed a) to examine the apoE4 allele frequency in definitive cases of spo-radic Alzheimer's disease and, in control brains show-ing plaque and tangle counts below the consensus AD
threshold; b) to determine if the apo E genotype dis-tribution varies in AD, AD/PD and PD.
SUMMARY OF THE INVENTION
One aim of the present invention is to have a classification of Alzheimer's disease for use in diagnostic, prognosis and selection of an appropriate treatment for the patient.
2111~03 In accordance with the present invention there is provided a method for the clinical determination of the risk for the late-onset of Alzheimer's disease of a patient, which comprises, determining the number of copies of the apo E gene allele E4 in a biological sample of said patient using an appropriate E4 probe, wherein zero, one or two copies of E4 indicates level of incidence of late-onset of Alzheimer's disease.
In accordance with the present invention there is provided a method for the prognosis for the treatment of Alzheimer's wherein one copy of E4 indicates a level of choline acetyltransferase reduced by half.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a distribution of E4 allele fre-quency by age in post-mortem Alzheimer's diseases and controlsi Fig. 2 shows a distribution of E4 allele fre-quency by age in post-mortem Alzheimer's diseases and controls; and Fig. 3 shows a distribution of E4 allele fre-quency by age in clinical (phenotype) and post-mortem (genotype) cases of Alzheimer's disease.
DETAILED DESCRIPTION OF THE lN~N~lION
Apolipoprotein E (apo E) is associated with Alzheimer's neurofibrillary tangles and ~-amyloid pro-tein in senile plaques. It also plays a critical role in the redistribution of lipids following differentia-tion and degeneration in the brain. Recent studies have shown high frequencies of the apo E4 allele in familial and sporadic cases of Alzheimer's disease (AD).
21115~
In accordance with the present invention, the apoE genotype was determined by allele-specific exten-sion of 113 post-mortem cases of sporadic AD and 77 age-matched control brains shown to be free of AD neu-ropathological feature, and then calculated the fre-quency of the various allelic forms of apoE (E2, E3, E4). Because Alzheimer's disease and Parkinson's dis-ease (PD) share several neuropathological characteris-tics, the apo E genotypes were also determined in 17 post-mortem cases of idiopathic Parkinson's disease and 19 cases with both Alzheimer's and Parkinson's dis-eases.
The overall frequency of the E4 allele in the AD cases was 33% compared to 5% in controls while that of E2 was 2% in AD versus 5% in controls and that of E3, 65% in AD versus 90% in controls (p<0.001). Of all the individuals examined in this study, 91% (10/11) of those with 2 copies of the E4 allele, and 90% (55/61) of those with one copy of the E4 allele were neuropa-thologically confirmed cases of AD. The frequencies ofthe E4 and E3 alleles were similar for AD and AD/PD
whereas the E4 allele was absent in PD without demen-tia.
A single copy of the E4 allele is sufficient to represent a major biological risk factor in sporadic AD.
Brain Tissue Samples Neuropathologically confirmed brains were obtained from the Douglas Hospital Brain Bank. Seventy seven controls (C) representing a random population and 113 sporadic AD frozen brains were used in this study.
Seventeen were PD without dementia and 19 with AD/PD.
Neuropathological confirmation of idiopathic Parkin-son's disease was based on the presence of Lewy Bodies ~ 5 ~ 2111~
and on the loss of pigmented neurons in the substantianigra pars compacta as described in Aubert et al. (19).
The diagnosis of AD~ was confirmed neuropathologically in all cases according to the criteria of Khachaturian et al. (20) as adapted by Aubert et al. (19); all other conditions were excluded.
ApoE genotype High molecular weight DNA was isolated from frozen cerebellum or temporal cortex as adapted from Goelz et al. (21). Apo E genotype was determined by allele-specific extension of purified brain DNA using a modification of the method of Main et al. (22). The primers labeled D,E,F,G, and H were synthesized for us by Genosys Biotech (The Woodlanf, TX); the primer sequences are given in Main et al. (22). Reactions were carried out in a volume of 50uL containing 1 ug of DNA;
deoxyadenosine triphosphate, deoxycytidine triphosph-ate, deoxythymidine triphosphate and deoxyguanosine triphosphate, each 0.2 mmol/L; 10% dimethyl sulfoxide;
12.5 pmol of either primer D,E,F,G,; 25 pmol of primer H; and 10 uL of 10 PCR reaction buffer (Vector Biosys-tem, Toronto, ONT.) The DNA in the reaction mixture was first dena-tured for 10 min. at 96C and then cooled to 4C. Oneunit of Taq polymerase (Vector Biosystem, Toronto, ONT) was then added to each sample. Each sample was reheated for 2 min. at 96C and subjected to 30 cycles in a thermal cycler with each cycle consisting of a 10 sec denaturation at 96C, 30 sec annealing at 58C and 1 min. extension at 65C. The reaction products were visualized by electrophoresis of 10uL of the reaction mixture in a 1% agarose gel containing TPE buffer (0.08 mol/L Tris-phosphate, 0.002 mol/L EDTA) and ethidium bromide (0.15 ug/mL) for 1 hr at 67v. The gel were then photographed and the banding profile was compared to known standards.
RESULTS
The ages of controls were matched with the patients' ages (Table 1).
Table 1 Apo E Genotype and Allele Frequency AL~llElhlER'S DISEASE CONTROLS Eastern All (n=113) M (n=~9) F(n=~4) ~l (n=77)M (n=53)F(n=24) Canadlans (n=1 02) Age (yr) 76.4 (9.3) 75.9(9.7) 76.9(9.0) 70.1 (15.2) 70.7 (13.9) 70.8(14.7) ~6.3(~.5) Genotype (%) E4/4 8.8~,10.2 7.4 1.3 0.0 4.0~ 3.9 E3/3 40.7~47.5 33.3 81.8 84.9 76.0~ 61.7 E212 0.9(~ 1.7 0.0 0.0 0.0 0.0 2 E413 46.9~37.3 57.4& 6.5 3.8 1~0~ 20.6 E412 1.7(~l) 1.7 1.9 0.0 0.0 0.0 9.8 E3/2 o.g~ 1.7 0.0 10.4 11.3 8.0~ 2 AJlele (Frequency)~
E4 0.33@~0.30 0.37 0.05 0.02 0.1- 0.152 E3 0.65~0.67 0.62 0.90 0.92 0.86 0.77 E2 0.02@0.03 0.01 0.05 0.06 0.04- 0.078 Mean (SD) for age @~ p<O.001 versus controls; ~ p<0.05 and p<0.001 versus men No""~ 1ernic populta~on from EastemCanada: ref ??
The apo E genotype distribution and allele fre-quencies are given in Table 1. The distribution of apo E phenotypes among a random population from eastern Canada as a comparison to the distribution of apo E
genotypes was determined (23). The E4 allele frequency is significantly higher (6-fold) in the Alzheimer's population. The genotypic distribution profile shows an enrichment of the E4/4 and E4/3 genotypes and a marked reduction of the E3/3 genotype in AD. Apo E4/3 genotype is markedly enriched in females versus males, in both AD and control cases. Of all the individuals examined in this study, 91% (10/11) of those with 2 copies of the E4 allele, and 90% (55/61) of those with one copy of the E4 allele were definitive cases of AD.
The neuropathological reports of the control individu-als carrying one or 2 copies of the E4 allele indicates the presence of plaques and tangles below the AD
threshold, but moderate to severe neuronal cell losses in Ammon's horn and subicular areas of the hippocampus.
Figure 1 shows the distribution of E4 allele frequency as a function of age in post-mortem tissues of AD and control individuals. An extended analysis of the genotype distribution in AD, AD/PD and PD in Figure 2 indicates that the frequency of the E4 and E3 alleles were similar in the AD and AD/PD groups whereas E4 allele was absent in PD.
The postmortem distribution of the E4 allele as a function of age in sporadic Alzheimer's disease is nearly identical the phenotypic distribution reported recently in clinically diagnosed Alzheimer's patients (Figure 3).
In previous clinical study up to 20% of the non-demented controls had at least one copy of the E4 allele and was hypothesized that a large proportion of the individuals would develop AD with time (13). Due to the complexity and logistical problems associated with a 10 to 15 year longitudinal study of the non-demented E4 controls, the alternative strategy of thecase-control study was described in this report. To examine the relationship between the E4 allele and the incidence of AD, genotypic analysis of apoE was carried out on samples of autopsied brains from individuals (age 43 to 95 years) with little or no neurodegenera-tive changes associated with AD.
The present results demonstrate a strong asso-ciation between the E4 allele and neuropathologically confirmed sporadic Alzheimer's disease. The overall frequency of the E4 allele in the AD cases was 33% com-pared to 5% in controls while that of E2 was 2% in ADversus 5% in controls and that of E3, 65% in AD versus 90% in controls. These frequencies are similar to those reported previously in sporadic AD and late onset familial AD (13,15). Furthermore, comparison of E4 allele frequency as a function of age in clinical and autopsied cases of sporadic AD shows a very similar distribution profile (13), supporting the diagnostic value of apo E phenotyping in living patients.
The present genotyping study has also revealed that 91% (10/11) of E4 homozygotes and 90% (55/61) of E4 heterozygotes had definitive (neuropathologically confirmed) Alzheimer's disease. Thus, the presence of a single copy of the E4 allele represents a major bio-logical risk factor for sporadic AD. Less than 10% of controls (n=77) carried the E4 allele. Although all controls had plaque and tangle counts below the AD
threshold level, the E4 allele carriers (1 homozygotes and 5 heterozygotes) demonstrated moderate to severe neuronal loss in the hippocampal Ammon's horn and subicular areas. Presumably, these individuals might represent an early phase (pre-symptomatic) of AD
pathophysiology. If this assumption proves to be cor-rect, the results of the present invention would clearly indicate that neuronal cell loss in the hip-pocampal area of E4 carriers precedes the formation of plaques and tangles.
Epidemiological studies have shown that risk of dementia in subjects suffering from Parkinson's disease is twice that of healthy controls: the overall cumula-tive probability of developing dementia in a period of 5 years is 21% in PD versus 5.7% in healthy controls (24). A common pathophysiology has been suggested by histopathological and neurochemical observations: the presence in cortical areas of demented parkinsonians of - ~llISQ3 plaques, tangles, neuronal cell losses (16,17) and the concomitant loss of cholinergic and serotoninergic activities (19,25). The present results do not support the concept of a common pathophysiology in AD and PD.
The distribution of apo E isoforms as determined by genotypic analysis in AD, AD/PD and PD highlights two distinct entities: the PD population in which E4 is virtually absent and the AD population which is highly enriched in E4. The remaining patients showing neuro-pathological changes consistent with both AD and PDdemonstrated an apoE isoform distribution that was very similar to AD, suggesting that the occurrence of PD in AD patients is an independent phenomenon in E4 carri-ers.
The immunological detection of apoE in plaques and tangles in AD and the enrichment of the E4 allele in AD suggest a fundamental role for apoE4 in the etiopathology of this disease. ApoE expression has been shown to be critical to the synaptic remodelling occur-ring in response to cell loss and differentiation in the CNS (2,3,6). In the normal aging human brain, the age-related decline in lipid levels and in cell number is apparently compensated by the active remodelling of neuronal pathways in an attempt to preserve the func-tional integrity of the CNS. Since synaptic and den-dritic remodelling of neurons requires the induction of apo E and apoE/apoB (LDL) receptor expression (3), it is conceivable that the presence of apoE4 in the CNS
may perturb cholesterol and phospholipid homoeostasis and interfere directly with synaptic plasticity. This could explain the marked decrease in presynaptic termi-nal density reported in cerebral cortex of AD, the syn-aptic losses in frontal and temporal cortices (26,27) and the poor synaptic plasticity reported in the hip-pocampus of AD (28,29,30,31,32).
211150~`
Incidence of late-onset Alzheimer's disease 2 copies of E4= >90%
l copy of E4= 70%
0 copy of E4= impossible to determine with precision While the invention has been described in con-nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia-tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
- 2~11503 REFERENCES
1 Elshourbagy NA, Liao WS, Mahley RW, Taylor JM
Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets. Proc.
Natl. Acad. Sci., 1985, 82: 203-207 ~
2 Poirier J, Hess M, May PC, Finch CE Apolipopro-tein E-and GFAP-RNA in hippocampus during reactive syn-aptogenesis and terminal proliferation. Mol. Brain Res., 1991, 11: 97-106.
BACKGROUND OF THE lNv~N~lION
Apolipoprotein E (apo E) is a well character-ized lipophilic protein associated with plasma and CSFlipoproteins. Apo E is synthesized primarily by the liver, but also at other sites including brain, macro-phages and adrenals (1). Furthermore, apo E is unique among apolipoproteins in that it is has a special rele-vance to the central and peripheral nervous systems.Apolipoprotein E (apoE) is important in modulating cholesterol and phospholipid transport from one cell to another. It is a key determinant in the cellular recognition and internalization of cholesterol-rich lipoproteins in the developing brain and in the response to neuronal injury (2,3,4,5). It also plays a fundamental role in the central nervous system (CNS) during synaptic remodelling induced by neuronal differentiation (2,3,6).
The apo E gene is located on chromosome 19, within a region which had previously been associated with familial late-onset cases of Alzheimer's disease (7). The structural gene for apo E is polymorphic, the three most common isoforms coded for are designated E2, E3 and E4 (5). These isoforms differ by amino acid substitutions at one or both of two sites, residues 112 and 158. The E2 isoform has Cys residues at sites 112 and 158. E3 has a Cys residue at site 112 and an Arg at site 158, and E4 has an Arg residue at sites 112 and 158. An individual thus may be homozygous (E2/2, E3/3 or E4/4) or heterozygous (E4/2, E4/3 or E3/2).
Apo E has been shown to be present in plaques and dystrophic neurites that characterize the neuropil of the Alzheimer's brain (8,9). Although apo E mRNA
and protein content is increased in response to neuro-~111503 nal cell loss in rat (2,3) and during demylination inhumans with multiple sclerosis (10), apo E expression remains relatively unchanged in the hippocampus of Alzheimer's (AD) patients (11) despite marked neuronal cell loss and differentiation in this structure (12).
Recent analysis of the apo E polymorphism in sporadic cases of Alzheimer's disease (13,14) has shown an increased frequency of the apo E4 allele in these indi-viduals. Moreover, there is a correlation between the age of onset for the disease and the number of copies of the E4 allele in that E4 homozygotes tend to have an earlier age of onset (13). Increased frequencies of the E4 allele have also been reported in familial cases of late onset AD (15).
Although the concept of subtypes of AD remains controversial to date,;it is clear that a significant portion of the AD population shows extrapyramidal symp-toms and neuropathological changes consistent with idiopathic Parkinson's disease (PD) (16, 17). The esti-mated prevalence of dementia in PD varies from 10% to 40% (18), the combination of AD/PD pathological fea-tures being common.
The present study was designed a) to examine the apoE4 allele frequency in definitive cases of spo-radic Alzheimer's disease and, in control brains show-ing plaque and tangle counts below the consensus AD
threshold; b) to determine if the apo E genotype dis-tribution varies in AD, AD/PD and PD.
SUMMARY OF THE INVENTION
One aim of the present invention is to have a classification of Alzheimer's disease for use in diagnostic, prognosis and selection of an appropriate treatment for the patient.
2111~03 In accordance with the present invention there is provided a method for the clinical determination of the risk for the late-onset of Alzheimer's disease of a patient, which comprises, determining the number of copies of the apo E gene allele E4 in a biological sample of said patient using an appropriate E4 probe, wherein zero, one or two copies of E4 indicates level of incidence of late-onset of Alzheimer's disease.
In accordance with the present invention there is provided a method for the prognosis for the treatment of Alzheimer's wherein one copy of E4 indicates a level of choline acetyltransferase reduced by half.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a distribution of E4 allele fre-quency by age in post-mortem Alzheimer's diseases and controlsi Fig. 2 shows a distribution of E4 allele fre-quency by age in post-mortem Alzheimer's diseases and controls; and Fig. 3 shows a distribution of E4 allele fre-quency by age in clinical (phenotype) and post-mortem (genotype) cases of Alzheimer's disease.
DETAILED DESCRIPTION OF THE lN~N~lION
Apolipoprotein E (apo E) is associated with Alzheimer's neurofibrillary tangles and ~-amyloid pro-tein in senile plaques. It also plays a critical role in the redistribution of lipids following differentia-tion and degeneration in the brain. Recent studies have shown high frequencies of the apo E4 allele in familial and sporadic cases of Alzheimer's disease (AD).
21115~
In accordance with the present invention, the apoE genotype was determined by allele-specific exten-sion of 113 post-mortem cases of sporadic AD and 77 age-matched control brains shown to be free of AD neu-ropathological feature, and then calculated the fre-quency of the various allelic forms of apoE (E2, E3, E4). Because Alzheimer's disease and Parkinson's dis-ease (PD) share several neuropathological characteris-tics, the apo E genotypes were also determined in 17 post-mortem cases of idiopathic Parkinson's disease and 19 cases with both Alzheimer's and Parkinson's dis-eases.
The overall frequency of the E4 allele in the AD cases was 33% compared to 5% in controls while that of E2 was 2% in AD versus 5% in controls and that of E3, 65% in AD versus 90% in controls (p<0.001). Of all the individuals examined in this study, 91% (10/11) of those with 2 copies of the E4 allele, and 90% (55/61) of those with one copy of the E4 allele were neuropa-thologically confirmed cases of AD. The frequencies ofthe E4 and E3 alleles were similar for AD and AD/PD
whereas the E4 allele was absent in PD without demen-tia.
A single copy of the E4 allele is sufficient to represent a major biological risk factor in sporadic AD.
Brain Tissue Samples Neuropathologically confirmed brains were obtained from the Douglas Hospital Brain Bank. Seventy seven controls (C) representing a random population and 113 sporadic AD frozen brains were used in this study.
Seventeen were PD without dementia and 19 with AD/PD.
Neuropathological confirmation of idiopathic Parkin-son's disease was based on the presence of Lewy Bodies ~ 5 ~ 2111~
and on the loss of pigmented neurons in the substantianigra pars compacta as described in Aubert et al. (19).
The diagnosis of AD~ was confirmed neuropathologically in all cases according to the criteria of Khachaturian et al. (20) as adapted by Aubert et al. (19); all other conditions were excluded.
ApoE genotype High molecular weight DNA was isolated from frozen cerebellum or temporal cortex as adapted from Goelz et al. (21). Apo E genotype was determined by allele-specific extension of purified brain DNA using a modification of the method of Main et al. (22). The primers labeled D,E,F,G, and H were synthesized for us by Genosys Biotech (The Woodlanf, TX); the primer sequences are given in Main et al. (22). Reactions were carried out in a volume of 50uL containing 1 ug of DNA;
deoxyadenosine triphosphate, deoxycytidine triphosph-ate, deoxythymidine triphosphate and deoxyguanosine triphosphate, each 0.2 mmol/L; 10% dimethyl sulfoxide;
12.5 pmol of either primer D,E,F,G,; 25 pmol of primer H; and 10 uL of 10 PCR reaction buffer (Vector Biosys-tem, Toronto, ONT.) The DNA in the reaction mixture was first dena-tured for 10 min. at 96C and then cooled to 4C. Oneunit of Taq polymerase (Vector Biosystem, Toronto, ONT) was then added to each sample. Each sample was reheated for 2 min. at 96C and subjected to 30 cycles in a thermal cycler with each cycle consisting of a 10 sec denaturation at 96C, 30 sec annealing at 58C and 1 min. extension at 65C. The reaction products were visualized by electrophoresis of 10uL of the reaction mixture in a 1% agarose gel containing TPE buffer (0.08 mol/L Tris-phosphate, 0.002 mol/L EDTA) and ethidium bromide (0.15 ug/mL) for 1 hr at 67v. The gel were then photographed and the banding profile was compared to known standards.
RESULTS
The ages of controls were matched with the patients' ages (Table 1).
Table 1 Apo E Genotype and Allele Frequency AL~llElhlER'S DISEASE CONTROLS Eastern All (n=113) M (n=~9) F(n=~4) ~l (n=77)M (n=53)F(n=24) Canadlans (n=1 02) Age (yr) 76.4 (9.3) 75.9(9.7) 76.9(9.0) 70.1 (15.2) 70.7 (13.9) 70.8(14.7) ~6.3(~.5) Genotype (%) E4/4 8.8~,10.2 7.4 1.3 0.0 4.0~ 3.9 E3/3 40.7~47.5 33.3 81.8 84.9 76.0~ 61.7 E212 0.9(~ 1.7 0.0 0.0 0.0 0.0 2 E413 46.9~37.3 57.4& 6.5 3.8 1~0~ 20.6 E412 1.7(~l) 1.7 1.9 0.0 0.0 0.0 9.8 E3/2 o.g~ 1.7 0.0 10.4 11.3 8.0~ 2 AJlele (Frequency)~
E4 0.33@~0.30 0.37 0.05 0.02 0.1- 0.152 E3 0.65~0.67 0.62 0.90 0.92 0.86 0.77 E2 0.02@0.03 0.01 0.05 0.06 0.04- 0.078 Mean (SD) for age @~ p<O.001 versus controls; ~ p<0.05 and p<0.001 versus men No""~ 1ernic populta~on from EastemCanada: ref ??
The apo E genotype distribution and allele fre-quencies are given in Table 1. The distribution of apo E phenotypes among a random population from eastern Canada as a comparison to the distribution of apo E
genotypes was determined (23). The E4 allele frequency is significantly higher (6-fold) in the Alzheimer's population. The genotypic distribution profile shows an enrichment of the E4/4 and E4/3 genotypes and a marked reduction of the E3/3 genotype in AD. Apo E4/3 genotype is markedly enriched in females versus males, in both AD and control cases. Of all the individuals examined in this study, 91% (10/11) of those with 2 copies of the E4 allele, and 90% (55/61) of those with one copy of the E4 allele were definitive cases of AD.
The neuropathological reports of the control individu-als carrying one or 2 copies of the E4 allele indicates the presence of plaques and tangles below the AD
threshold, but moderate to severe neuronal cell losses in Ammon's horn and subicular areas of the hippocampus.
Figure 1 shows the distribution of E4 allele frequency as a function of age in post-mortem tissues of AD and control individuals. An extended analysis of the genotype distribution in AD, AD/PD and PD in Figure 2 indicates that the frequency of the E4 and E3 alleles were similar in the AD and AD/PD groups whereas E4 allele was absent in PD.
The postmortem distribution of the E4 allele as a function of age in sporadic Alzheimer's disease is nearly identical the phenotypic distribution reported recently in clinically diagnosed Alzheimer's patients (Figure 3).
In previous clinical study up to 20% of the non-demented controls had at least one copy of the E4 allele and was hypothesized that a large proportion of the individuals would develop AD with time (13). Due to the complexity and logistical problems associated with a 10 to 15 year longitudinal study of the non-demented E4 controls, the alternative strategy of thecase-control study was described in this report. To examine the relationship between the E4 allele and the incidence of AD, genotypic analysis of apoE was carried out on samples of autopsied brains from individuals (age 43 to 95 years) with little or no neurodegenera-tive changes associated with AD.
The present results demonstrate a strong asso-ciation between the E4 allele and neuropathologically confirmed sporadic Alzheimer's disease. The overall frequency of the E4 allele in the AD cases was 33% com-pared to 5% in controls while that of E2 was 2% in ADversus 5% in controls and that of E3, 65% in AD versus 90% in controls. These frequencies are similar to those reported previously in sporadic AD and late onset familial AD (13,15). Furthermore, comparison of E4 allele frequency as a function of age in clinical and autopsied cases of sporadic AD shows a very similar distribution profile (13), supporting the diagnostic value of apo E phenotyping in living patients.
The present genotyping study has also revealed that 91% (10/11) of E4 homozygotes and 90% (55/61) of E4 heterozygotes had definitive (neuropathologically confirmed) Alzheimer's disease. Thus, the presence of a single copy of the E4 allele represents a major bio-logical risk factor for sporadic AD. Less than 10% of controls (n=77) carried the E4 allele. Although all controls had plaque and tangle counts below the AD
threshold level, the E4 allele carriers (1 homozygotes and 5 heterozygotes) demonstrated moderate to severe neuronal loss in the hippocampal Ammon's horn and subicular areas. Presumably, these individuals might represent an early phase (pre-symptomatic) of AD
pathophysiology. If this assumption proves to be cor-rect, the results of the present invention would clearly indicate that neuronal cell loss in the hip-pocampal area of E4 carriers precedes the formation of plaques and tangles.
Epidemiological studies have shown that risk of dementia in subjects suffering from Parkinson's disease is twice that of healthy controls: the overall cumula-tive probability of developing dementia in a period of 5 years is 21% in PD versus 5.7% in healthy controls (24). A common pathophysiology has been suggested by histopathological and neurochemical observations: the presence in cortical areas of demented parkinsonians of - ~llISQ3 plaques, tangles, neuronal cell losses (16,17) and the concomitant loss of cholinergic and serotoninergic activities (19,25). The present results do not support the concept of a common pathophysiology in AD and PD.
The distribution of apo E isoforms as determined by genotypic analysis in AD, AD/PD and PD highlights two distinct entities: the PD population in which E4 is virtually absent and the AD population which is highly enriched in E4. The remaining patients showing neuro-pathological changes consistent with both AD and PDdemonstrated an apoE isoform distribution that was very similar to AD, suggesting that the occurrence of PD in AD patients is an independent phenomenon in E4 carri-ers.
The immunological detection of apoE in plaques and tangles in AD and the enrichment of the E4 allele in AD suggest a fundamental role for apoE4 in the etiopathology of this disease. ApoE expression has been shown to be critical to the synaptic remodelling occur-ring in response to cell loss and differentiation in the CNS (2,3,6). In the normal aging human brain, the age-related decline in lipid levels and in cell number is apparently compensated by the active remodelling of neuronal pathways in an attempt to preserve the func-tional integrity of the CNS. Since synaptic and den-dritic remodelling of neurons requires the induction of apo E and apoE/apoB (LDL) receptor expression (3), it is conceivable that the presence of apoE4 in the CNS
may perturb cholesterol and phospholipid homoeostasis and interfere directly with synaptic plasticity. This could explain the marked decrease in presynaptic termi-nal density reported in cerebral cortex of AD, the syn-aptic losses in frontal and temporal cortices (26,27) and the poor synaptic plasticity reported in the hip-pocampus of AD (28,29,30,31,32).
211150~`
Incidence of late-onset Alzheimer's disease 2 copies of E4= >90%
l copy of E4= 70%
0 copy of E4= impossible to determine with precision While the invention has been described in con-nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia-tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
- 2~11503 REFERENCES
1 Elshourbagy NA, Liao WS, Mahley RW, Taylor JM
Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets. Proc.
Natl. Acad. Sci., 1985, 82: 203-207 ~
2 Poirier J, Hess M, May PC, Finch CE Apolipopro-tein E-and GFAP-RNA in hippocampus during reactive syn-aptogenesis and terminal proliferation. Mol. Brain Res., 1991, 11: 97-106.
3 Poirier J, Baccichet A, Dea D, Gauthier S Role of Hippocampal Cholesterol Synthesis and Uptake During Reactive Synaptogenesis in Adult Rats, Neuroscience, 1993, 55: 81-90.
4 Boyle JK, Zoellner CD, Anderson LJ, Kosick LM, Pitas RE, Weisgraber KH , Hui DY, Malhey RW, Gebicke-Haeter PJ, Ignatius MJ, Shooter EM. A role for apol-ipoprotein E, apolipoprotein A-l, and low density lipo-protein receptors in cholesterol transport during regeneration and remyelination of rat sciatic nerve. J
Clin. Invest., 1989, 83: 1015-1031.
Mahley RW Apolipoprotein E: cholesterol trans-port protein with expanding role in cell biology. Sci-ence, 1988, 240: 622-630.
6 Poirier J, Hess M, May PC, Finch CE Cloning of hippocampal poly(A)RNA sequences that increase after entorhinal cortex lesion in adult rat., Mol. Brain Res., 1991, 9: 191-195.
- 12 - 21115~3 7 Schellenberg GD, Boehnke M., Wijsman EM Genetic association and linkage analysis o~ the apolipoprotein CII locus and familial Alzheimer's disease., Ann. Neurol., 1992, 31: 223-227 8 Namba Y, Tomonaga M, Kawasaki H. Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer's disease. Brain Res., 1991, 541: 163-166.
9 Wisniewski T, Frangione B, Apolipoprotein E: a pathological chaperon protein in patients with cerebral and systemic amyloid, Neurosci. Lett., 1991, 135: 235-238 Rifai N, Christénson RH, Gelman BB, Silverman LM
Changes in cerebrovascular fluid apolipoprotein E index in patients with multiple sclerosis during demyelina-tion and remyelination, Clin. Chem., 1987, 33:1155-1157.
11 Poirier J, Aubert I, Bertrand P, Dea D, Quirion R, Nalbantoglu J, Gilfix BG, Hippocampal Apolipoprotein E4, Amyloid Plaques ~and Cholinergic Dysfunction in Alzheimer's disease. Nature, date unknown.
12 Van Hoesen GW, Hyman BT, Damasio AR Entorhinal cortex pathology in Alzheimer's disease, Hippocampus, 1991, 1:1-8.
13 Poirier J, Davignon J, Bouthillier D, Kogan S, Bertrand P, Gauthier S Apolipoprotein E phenotype and Alzheimer's Disease, Lancet, 1993, 342: 697-699.
14 Saunders AM, Strittmatter WJ, Schmechel D, St-George-Hyslop PH, Pericak-Vance MA, Joo SH, Rosi BA, Gusella JF, MacClachlan DR, Alberts MJ, Hulette C, - 13 - 2111~0~
Crain B, Goldgaber D, Roses AD. Association of apolipo-protein E allele _4 with late onset familial and spo-radic Alzheimer's disease. Neurology, 1993, 43:1467-1472.
Corder EH, Saunders AM, Strittmatter WJ, Sch-mechel DE, Gaskell PC, Small GW, Roses AD, Pericak-Vance MA, Gene dose of apolipoprotein E type 4 and risk of Alzheimer's disease in late onset families, Science, 1993, 261:921-923.
16 Boller F, Muzutani T, Roessmann U, Gambetti P
Parkinson's disease, dementia and Alzheimer's disease:
a clinicopathological correlation., A~n Neurol. , 1980, 7:329-335.
17 Ditter SM, Mirra SS, Neuropathological and clinical features of Parkinson's disease in Alzheimer's disease patients., Neurology, 1987, 37:754-760.
18 Mayeux R, Stern Y, Rosenstein R, Marder K, Hauser A, Cote L, Fahn S An estimate of the prevalence of dementia in idiopathic Parkinson's disease., Arch.
Neurol., 1988: 45:260-262.
19 Aubert I, Araujo DM, Cecyre D, Robitaille Y, Gauthier S, Quirion R Comparative alterations of nico-tinic and muscarinic binding sites in Alzheimer's and Parkinson's diseases., J. Neurochem., 1992, 58:529-541.
Khachaturian ZS Diagnosis of Alzheimer's dis-ease., Arch.Neurol., 1985, 42:1097-1105.
21 Goelz SE, Hamilton SR, Vogelstein B, Purifica-tion of DNA from paraffin embedded human tissues., Bio-chem.Biophys.Res.Comm., 1985, 130: 118-126.
21115~
Z2 Main RF, Jones PJH, McGillivery RTA, Banfield DK, Apolipoprotein E genotyping using the polymerase chain reaction and allele specific oligonucleotide primers., J.LipidRes., 1991, 32:183-187.
23 Sing CF, Davignon J Role of apolipoprotein E
polymorphism in determining normal plasma lipid and lipoprotein variation., Am J. Hum. Genet., 1985, 37:268-278.
24 Ditter SM, Mirra SS Neuropathologic and clinical features of Parkinson's disease in Alzheimer's disease patients., Neurology, 1987, 37:754-760.
D'Amato RJ, Zweig RM, Whitehouse PJ, Wenk GL, Singer HD, Mayeux R, Price DL, Snyder SH., Aminergic systems in Alzheimer's disease and Parkinson's dis-ease., Ann. Neurol., 1987, 22:229-238.
26 Masliah E, Terry RD, DeTeresa RM. Immunohisto-chemical quantification of synapse-related protein syn-aptophysin in Alzheimer's disease., Neurosci. Le~. , 1989, 103: 234-239.
27 Davies CA, Mann DMA Sumpter PQ. Quantitative morphometric analysis of the neuronal and synaptic con-tent of the frontal and temporal cortex in patients with Alzheimer's disease., J. Neurol. Sci., 1987, 78: 151-164.
28 deRuiter JP, Uylings HBM, Morphometric and den-dritic analysis of fascia dentata granule cells in human aging and in senile dementia., Brain Res., 1987, 402: 217-229.
21115~3 29 Ransmayr G, Cervera P, Hirsch E, Agid Y. Choline acetyl transferase-like immunoreactivity in the hip-pocampal formation of controls subjects and patients with Alzheimer's disease, Neuroscience, 1989, 32: 701-714.
Represa A, Duyckerts C, Tremblay E., Is senile dementia of the Alzheimer type associated with hip-pocampal plasticity?, BrainRes., 1988, 457: 355-359.
31 Flood DG, Coleman PD, Failed compensatory den-dritic growth as a pathophysiological process in Alzheimer's disease., Can.J.Neurol. Sci., 1986, 13: 475-479.
32 Honer WG, Dickson DW, Gleeson J. Regional synap-tic pathology in Alzheimer's disease., Neurobiol. Aging, 1992, 13: 375-382.
Clin. Invest., 1989, 83: 1015-1031.
Mahley RW Apolipoprotein E: cholesterol trans-port protein with expanding role in cell biology. Sci-ence, 1988, 240: 622-630.
6 Poirier J, Hess M, May PC, Finch CE Cloning of hippocampal poly(A)RNA sequences that increase after entorhinal cortex lesion in adult rat., Mol. Brain Res., 1991, 9: 191-195.
- 12 - 21115~3 7 Schellenberg GD, Boehnke M., Wijsman EM Genetic association and linkage analysis o~ the apolipoprotein CII locus and familial Alzheimer's disease., Ann. Neurol., 1992, 31: 223-227 8 Namba Y, Tomonaga M, Kawasaki H. Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer's disease. Brain Res., 1991, 541: 163-166.
9 Wisniewski T, Frangione B, Apolipoprotein E: a pathological chaperon protein in patients with cerebral and systemic amyloid, Neurosci. Lett., 1991, 135: 235-238 Rifai N, Christénson RH, Gelman BB, Silverman LM
Changes in cerebrovascular fluid apolipoprotein E index in patients with multiple sclerosis during demyelina-tion and remyelination, Clin. Chem., 1987, 33:1155-1157.
11 Poirier J, Aubert I, Bertrand P, Dea D, Quirion R, Nalbantoglu J, Gilfix BG, Hippocampal Apolipoprotein E4, Amyloid Plaques ~and Cholinergic Dysfunction in Alzheimer's disease. Nature, date unknown.
12 Van Hoesen GW, Hyman BT, Damasio AR Entorhinal cortex pathology in Alzheimer's disease, Hippocampus, 1991, 1:1-8.
13 Poirier J, Davignon J, Bouthillier D, Kogan S, Bertrand P, Gauthier S Apolipoprotein E phenotype and Alzheimer's Disease, Lancet, 1993, 342: 697-699.
14 Saunders AM, Strittmatter WJ, Schmechel D, St-George-Hyslop PH, Pericak-Vance MA, Joo SH, Rosi BA, Gusella JF, MacClachlan DR, Alberts MJ, Hulette C, - 13 - 2111~0~
Crain B, Goldgaber D, Roses AD. Association of apolipo-protein E allele _4 with late onset familial and spo-radic Alzheimer's disease. Neurology, 1993, 43:1467-1472.
Corder EH, Saunders AM, Strittmatter WJ, Sch-mechel DE, Gaskell PC, Small GW, Roses AD, Pericak-Vance MA, Gene dose of apolipoprotein E type 4 and risk of Alzheimer's disease in late onset families, Science, 1993, 261:921-923.
16 Boller F, Muzutani T, Roessmann U, Gambetti P
Parkinson's disease, dementia and Alzheimer's disease:
a clinicopathological correlation., A~n Neurol. , 1980, 7:329-335.
17 Ditter SM, Mirra SS, Neuropathological and clinical features of Parkinson's disease in Alzheimer's disease patients., Neurology, 1987, 37:754-760.
18 Mayeux R, Stern Y, Rosenstein R, Marder K, Hauser A, Cote L, Fahn S An estimate of the prevalence of dementia in idiopathic Parkinson's disease., Arch.
Neurol., 1988: 45:260-262.
19 Aubert I, Araujo DM, Cecyre D, Robitaille Y, Gauthier S, Quirion R Comparative alterations of nico-tinic and muscarinic binding sites in Alzheimer's and Parkinson's diseases., J. Neurochem., 1992, 58:529-541.
Khachaturian ZS Diagnosis of Alzheimer's dis-ease., Arch.Neurol., 1985, 42:1097-1105.
21 Goelz SE, Hamilton SR, Vogelstein B, Purifica-tion of DNA from paraffin embedded human tissues., Bio-chem.Biophys.Res.Comm., 1985, 130: 118-126.
21115~
Z2 Main RF, Jones PJH, McGillivery RTA, Banfield DK, Apolipoprotein E genotyping using the polymerase chain reaction and allele specific oligonucleotide primers., J.LipidRes., 1991, 32:183-187.
23 Sing CF, Davignon J Role of apolipoprotein E
polymorphism in determining normal plasma lipid and lipoprotein variation., Am J. Hum. Genet., 1985, 37:268-278.
24 Ditter SM, Mirra SS Neuropathologic and clinical features of Parkinson's disease in Alzheimer's disease patients., Neurology, 1987, 37:754-760.
D'Amato RJ, Zweig RM, Whitehouse PJ, Wenk GL, Singer HD, Mayeux R, Price DL, Snyder SH., Aminergic systems in Alzheimer's disease and Parkinson's dis-ease., Ann. Neurol., 1987, 22:229-238.
26 Masliah E, Terry RD, DeTeresa RM. Immunohisto-chemical quantification of synapse-related protein syn-aptophysin in Alzheimer's disease., Neurosci. Le~. , 1989, 103: 234-239.
27 Davies CA, Mann DMA Sumpter PQ. Quantitative morphometric analysis of the neuronal and synaptic con-tent of the frontal and temporal cortex in patients with Alzheimer's disease., J. Neurol. Sci., 1987, 78: 151-164.
28 deRuiter JP, Uylings HBM, Morphometric and den-dritic analysis of fascia dentata granule cells in human aging and in senile dementia., Brain Res., 1987, 402: 217-229.
21115~3 29 Ransmayr G, Cervera P, Hirsch E, Agid Y. Choline acetyl transferase-like immunoreactivity in the hip-pocampal formation of controls subjects and patients with Alzheimer's disease, Neuroscience, 1989, 32: 701-714.
Represa A, Duyckerts C, Tremblay E., Is senile dementia of the Alzheimer type associated with hip-pocampal plasticity?, BrainRes., 1988, 457: 355-359.
31 Flood DG, Coleman PD, Failed compensatory den-dritic growth as a pathophysiological process in Alzheimer's disease., Can.J.Neurol. Sci., 1986, 13: 475-479.
32 Honer WG, Dickson DW, Gleeson J. Regional synap-tic pathology in Alzheimer's disease., Neurobiol. Aging, 1992, 13: 375-382.
Claims (2)
1. A method for the clinical determination of the risk for the late-onset of Alzheimer's disease of a patient, which comprises, determining the number of copies of the apo E gene allele E4 in a biological sample of said patient using an appropriate E4 probe, wherein zero, one or two copies of E4 indicates level of incidence of late-onset of Alzheimer's disease.
2. The method of claim 1, wherein one copy of E4 indicates the prognosis for the treatment of Alzheimer's with a level of choline acetyltransferase reduced by half.
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| PCT/CA1994/000681 WO1995016791A1 (en) | 1993-12-15 | 1994-12-13 | Apolipoprotein e polymorphism and alzheimer's disease |
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| US6022683A (en) | 1996-12-16 | 2000-02-08 | Nova Molecular Inc. | Methods for assessing the prognosis of a patient with a neurodegenerative disease |
| US5767337A (en) * | 1995-07-31 | 1998-06-16 | Duke University | Creation of human apolipoprotein E isoform specific transgenic mice in apolipoprotein deficient "knockout" mice |
| US6136530A (en) * | 1995-11-29 | 2000-10-24 | Texas Tech University Health Sciences Center | Compositions and methods for assessing risk factors in Alzheimer's disease |
| US6711551B1 (en) | 1996-06-05 | 2004-03-23 | Netzero, Inc. | Information provider, terminal and system and recording medium for the terminal |
| ES2115553B1 (en) * | 1996-10-23 | 1999-02-16 | Boehringer Ingelheim Espana | METHOD TO DETECT THE PRESENCE OF DNA POLYMORPHISMS, TEST KIT AND CORRESPONDING RECOMBINANT NUCLEIC ACIDS. |
| ES2128265B1 (en) * | 1997-06-04 | 2000-03-01 | Euroespes S A | GENETIC KIT FOR THE MOLECULAR CHARACTERIZATION OF ALZHEIMER'S DISEASE. |
| FR2765591B1 (en) * | 1997-07-01 | 2002-08-09 | Pasteur Institut | METHOD FOR DIAGNOSING ALZHEIMER'S DISEASE |
| ES2142248B1 (en) * | 1997-11-20 | 2000-11-16 | Univ Madrid Autonoma | POLYMORPHISMS IN THE PROMOTER OF THE HUMAN GENE OF APOLIPOPROTEIN E, AND ITS USES FOR DIAGNOSTIC AND THERAPEUTIC APPLICATIONS. |
| US6251587B1 (en) | 1997-12-16 | 2001-06-26 | Nova Molecular, Inc. | Method for determining the prognosis of a patient with a neurological disease |
| DE19855469C2 (en) * | 1998-12-01 | 2001-01-11 | Michael Esrich | Method for determining the apolipoprotein E genotype in a human sample |
| CA2409774A1 (en) * | 2000-05-23 | 2001-11-29 | Variagenics, Inc. | Methods for genetic analysis of dna to detect sequence variances |
| US7435541B2 (en) | 2000-05-23 | 2008-10-14 | Sequenom, Inc. | Restriction enzyme genotyping |
| CA2527588A1 (en) * | 2002-06-27 | 2004-01-08 | Aarhus Universitet | Disease risk estimating method using sequence polymorphisms in a specific region of chromosome 19 |
| ATE435302T1 (en) * | 2002-08-07 | 2009-07-15 | Novartis Pharma Gmbh | METHOD FOR PREDICTING TREATMENT SUCCESS WITH RIVASTIGMINE BASED ON DETERMINATION OF THE APOE GENOTYPE OF A DEMENTIA PERSON |
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| EP3931349B8 (en) * | 2019-02-28 | 2025-01-15 | The Rockefeller University | Apoe genotyping in cancer prognostics and treatment |
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