WO1996002670A1

WO1996002670A1 - Methods of prognosing the course and degree of dementia in patients with beta-amyloid-related diseases

Info

Publication number: WO1996002670A1
Application number: PCT/EP1995/002823
Authority: WO
Inventors: Maureen Claire Royston; Gareth Wyn Roberts; John Hardy
Original assignee: Smithkline Beecham Plc; University Of South Florida
Priority date: 1994-07-20
Filing date: 1995-07-12
Publication date: 1996-02-01
Also published as: GB9414624D0; AU3114295A

Abstract

A method of prognosing in a subject for the course and nature of disease progression and the ultimate degree of dementia which a subject would suffer during a β amyloid-related disease, wherein the presence of an ApoE2 isoform or DNA in a subject at risk of such disease or suspected of having such a disease or in the early stages of such a disease indicates that the subject is likely to have a milder, more benign course of disease with the extent of the eventual cognitive impairment or dementia being of mild or moderate degree, the method comprising detecting the presence or absence of ApoE isoforms or of DNA encoding ApoE isoforms in the subject.

Description

METHODS OF PROGNOSING THE COURSE AND DEGREE OF DEMENTIA IN PATIENTS WITH BETA-AMYLOID-RELATED DISEASES

The present invention relates to methods of prognosing the course and degree of dementia in patients with β amyloid-related disease, in particular Alzheimer-type 5 disease in individuals with Down's syndrome. β amyloid protein-related disease is a heterogeneous class of disorders characteristd by the deposition within the brain of insoluble deposits of the β amyloid protein (1). The eventual consequence of substantial numbers of β amyloid deposits is the emergence of a clinical syndrome of cognitive decline and increasing dementia. Such deposits have been shown to be present in a number of dementing syndromes and these include Alzheimer's disease, conical Lewy body disease, Parkinson's disease and the Alzheimer-type disease in patients with Down's syndrome. In addition β amyloid deposits are present in the brains of patients with vascular and cerebrovascular disease (2) and these latter conditions can predispose or contribute to the above diseases ( 1 ).

Recently, the apolipoprotein E (ApoE) genotype has been shown to be an important determinant in the etiology of AD (3,4,5,6,7,8,9,10) with the presence and number of E4 alleles being associated with increased risk and earlier ages of onset of disease in both familial cases linked to chromosome 19 and sporadic cases. The presence of E2 alleles has been claimed to decrease the risk (be 'protective') of late onset Alzheimer disease (5, 6, 11). This inference is based on the increased frequency of ApoE4 alleles in patients known to have Alzheimer's disease and the later age at onset of disease in patients with the ApoE2/3 genotype compared to the ApoE4/4 genotype (11,12). Such general 'protective' effects of the E2 allele have been reported previously in the general population with respect to heart disease (13, 14).

The exact role of ApoE in the pathology of Alzheimer-type disease is uncertain. ApoE is co- localised with β amyloid within plaques in the central nervous system (CNS) and has been shown to bind to β amyloid in vitro and not to tau proteins (15). This has led to the hypothesis that ApoE/tau interactions are critical in the pathophysiology of tangle formation and thus central to the process of Alzheimer-type diseases (15). However, neither parkin son dementia complex of guam nor aged Down's syndrome patients show increased levels of ApoE4 alleles despite the presence of large numbers of tangles in the CNS (16,17). As such the role of ApoE in the pathology of Alzheimer type dementia remains obscure.

At present there is no published data to document the role of ApoE genotype on the expression or degree of the clinical symptoms of the disease.

Methods of diagnosing or prognosing Alzheimer's disease have been described (WO 94/09155) based upon detecting (directly or indirectly) the presence or absence of an apolipoprotein E type 4 (ApoE4) isoform in the subject. The ApoE alleles E2, E3 and E4 are described in the literature (18,19).

However, at present there is no published data to document the role of ApoE genotype on the expression or degree of the clinical symptoms of the disease. Neuropathological studies have shown that all individuals with Downs syndrome (DS)(trisomy 21) develop the pathological hallmarks of Alzheimer's disease (AD) i.e. β amyloid protein deposition, neurofibrillary tangle formation and neuronal cell loss (2,20,21). The extent of this pathology varies from patient to patient for reasons which are unknown and the extent of pathology in a given patient is unpredictable (22,23,24,25,26). Frequent clinical anecdotes have suggested that the occurrence of dementia in elderly cases of DS is not universal and that the degree of dementia can vary substantially (22,23,24,25). In this respect the variability seen in patients with Downs syndrome parallels the clinical heterogeneity of Alzheimer's disease. However, recent studies have indicated that the development of such pathology and the associated dementia are significantly correlated to increasing age in the patient (27).

The occurrence of AD in DS (trisomy 21) is usually ascribed to the effects of overproduction of β amyloid caused by a gene dosage effect on the amyloid precursor protein (APP) carried on chromosome 21. Mutations in this gene, some of which lead to overproduction of β amyloid from APP, arc one cause of AD (1). However, it has recently become clear that the ApoE genotype is not only important in the etiology of late onset AD (3,4,5,6,7,8,9,10), but also modulates the age of onset of disease in a family in which the primary defect is a mutation which causes increased β amyloid production (12). In this family E4 allele inheritance also affected the age of onset such that E4 homozygotes had a lower age of onset than E3 homozygotes and E2 heterozygotes had a higher onset age.

Preliminary studies (17) are suggestive that the E2 allele might be associated with longevity in Down syndrome as it is in the general population. Again it is possible that this refers to the aforementioned general E2 protective effect against heart disease and late onset AD. However, in Down syndrome it has also been found that, contrary to the predisposing effect of the E4 allele in the development of AD- type dementia in non-DS individuals, there is no discernable increase in E4 allele frequency (despite the universal presence of Alzheimer-type pathology in the ens) in DS individuals (17). Thus, on the basis of the above data it would be predicted that in patients with

Downs syndrome increasing age will be linked to increasing degrees of Alzheimer-type pathology and these in turn would be associated with an increasing degree of dementia. Thus, given the association ofE2 with longevity it would be predicted that Down's patient with one or more E2 alleles would live longer, accumulate more Alzheimer-type pathology and thus have a more profound degree of dementia.

The present invention provides a method of prognosing in a subject for the course and nature of disease progression and the ultimate degree of dementia which a subject would suffer during a β amyloid-related disease, wherein the presence of an ApoE2 isoform or DNA in a subject at risk of such disease or suspected of having such a disease or in the early stages of such a disease indicates that the subject is likely to have a milder, more benign course of disease with the extent of the eventual cognitive impairment or dementia being of mild or moderate degree, the method comprising detecting the presence or absence of ApoE isoforms or of DNA encoding ApoE isoforms in the subject.

Suitable subjects include those which have not previously been diagnosed as afflicted with β amyloid-related disease and which have previously been determined to be at risk of developing β amyloid-related disease, in particular DS individuals. The presence of E2/E2 homozygote or E2/E3 heterozygote has been found to predict a milder, less dementing course in patients with Downs syndrome. By inference E3/E3 homozygotes or E4 heterozygotes might be expected to have a more severe course and degree of dementia.

The method of the invention is applicable to patients at risk of, suffering fron. or suspected of suffering from β amyloid-related diseases such as Alzheimer's disease, cortical Lewy body disease, Parkinson's disease and patients with vascular and cerebrovascular disease which predispose to the above diseases.

The step of detecting the presence or absence of ApoE isoforms or of DNA encoding such isoforms may be carried out either directly or indirectly by any suitable means, such as by techniques well known in the an, and is preferably carried out ex vivo (eg by means of the method described in 28). All generally involve the step of collecting a sample of biological material containing either DNA or ApoE from the subject, and then detecting which isoforms the subject possesses from that sample. For example, the detecting step may be carried out by collecting an ApoE sample from the subject (for example, from cerebrospinal fluid, or any other fluid or tissue containing ApoE), and then determining the presence or absence of an ApoE isoform in the ApoE sample (eg, by isoelectric focusing or immunoassay). In the alternative, the detecting step may be carried out by collecting a biological sample containing DNA from the subject, and then determining the presence or absence of DNA encoding an ApoE isoform in the biological sample. Any biological sample which contains the DNA of that subject may be employed, including tissue samples and blood samples, with blood cells being a particularly convenient source. Determining the presence or absence of DNA encoding an ApoE isoform may be carried out with an oligonucleotide probe labelled with a suitable detectable group, or by means of an amplification reaction such as a polymerase chain reaction or ligase chain reaction (the product of which amplification reaction may then be detected with a labelled oligonucleotide probe). Further, the detecting step may include the step of detecting whether the subject is heterozygous or homozygous for the gene encoding an ApoE isoform. Numerous different oligonucleotide probe assay formats are known which may be employed to carry out the present invention. Suitable examples of techniques and strategies for detecting the ApoE isoforms and encoding DNA are described in WO 94/09155.

It will be readily appreciated that the detecting steps may be carried out directly or indirectly. Thus, for example, any of the techniques described above for detecting ApoE2 may instead be used to detect ApoE3 and ApoE4. If either ApoE4 or ApoE3 is also detected in the subject, then it is determined that the subject is not homozygous for ApoE2; and if both ApoE4 and ApoE3 are detected in the subject, then it is determined that the subject is neither homozygous nor heterozygous for ApoE2.

Such prognostications will have considerable utility in the design, planning and implementation of clinical care for such patients. The method of the invention may be used to determine the appropriate therapeutic intervention or the degree of hospital social intervention or support required by a patient, by enabling prognostication of the rate of progression of dementia and the degree of dementia. Such prognostications will be of use in, for example, interpreting and deciding on the efficacy or likely outcome of clinical treatments for the above diseases; potential for response to different types of therapeutic intervention; selection of patients for clinical trials; analysis of data from clinical trials; forecasting and planning the requirements for facilities required to care for patients with such disease; and may be used to sub-divide and categorise such patients in order to maximise the use of such facilities and resources.

In a further aspect, the invention provides a method of treatment of β amyloid-related disease by the supplementation of ApoE2 protein expression, for example by direct administration of ApoE2 isoform or by gene therapy, or by the generation of an agonist which mimics the action of ApoE2 on the ApoE receptors. Direct adminstration may be by injection of the protein. Gene therapy may be by way of delivery of the ApoE2 gene or by enhancement of the expression of the patient's own ApoE2 gene. Such methods are generally known in the art.

EXAMPLE 1

The ApoE genotype was examined in a series of 10 clinically assessed elderly cases of DS. The case notes of each patient had been retrospectively assessed to rate the degree of dementia (Table 1). ApoE genotyping was carried out using a modification of standard procedures (24).

It was observed that the majority of the elderly cases of DS who had little or no dementia (Grade 1) had an E2 allele. In contrast to our expectations, such cases survived for longer (-E2 allele average age 58.2 years, +E2 allele average age 65.2 years), being on average 7 years older when they died.

TABLE 1

Age Gender ApoE Dementia Notes Genotype Score *

56 M 3 4 3 Bronchopneumonia

58 M 3 3 3 Bronchopneumonia

58 F 3 3 3 Bronchopneumonia

59 M 3 3 3 Bronchopneumonia

60 F 3 3 3 Bronchopneumonia

61 F 3 2 1 Thrombotic thrombocytopaenia

64 M 3 2 1 Bronchopneumonia

65 M 3 3 3 Bronchopneumonia

71 M 3 2 1 Bronchopneumonia

0 no dementia

1 mild dementia

2 moderate dementia

3 severe dementia for details of rating see ref 27

EXAMPLE 2

In a larger study involving 22 cases of patients with Downs syndrome a statistical analysis of the effects of E2 allelle status on the age at death and presence of dementia was undertaken . DS cases were collected at two sites: Manchester and Newcastle upon Type (UK).

Retrospective clinical assessment of all the cases from Manchester and the cases from Newcastle was made by analysis of the clinical notes to determine the presence or absence of a dementia state in the patients with Downs syndrome. The Clinical assessments were carried out blind to the genotyping results.

ApoE genotyping was carried out using a modification of standard procedures (24). Comparison of the genotypes with the clinical features of the disease in those

DS cases who lived to greater than 50 years (Table 2) revealed that, although the overall allele frequencies were within the published range for the ApoE locus, there were no E4 homozygotes at all within this selected data set and no E4 heterozygotes lived passed the age of 60 years. Of the patients with one E4 allele mean age at death was 57 years (n=8). Patients with an E3/E3 genotype had a mean age of death of 60 years. This difference suggests a trend whereby the effects of the E3/E4 and E3/E3 genotypes can be distinguished. In contradistinction, of the 7 individuals who lived passed the age of 60, 4 were E3/E2 heterozygotes and 1 was an E2 homozygote. Furthermore, all the E4 heterozygotes in the data set died with profound dementia as did the majority of the E3 homozygotes. In contrast, of those with an E2 allele, only 1 died with severe dementia (E2/3).

T ese data indicate that in cases where it is likely that the primary lesion (with regard to AD) is β amyloid-related, the ApoE2 genotype is of great importance in determining the clinical course and outcome in patients with AD and AD related dementias. It has previously been hypothesised that ApoE4 predisposes to the disease. The present invention is based upon the finding that ApoE2 status is associated with a milder form of the dementing illness relative to ApoE4.

STATISTICAL ANALYSIS Data was analysed to determine whether possession of an E2 allele had a significant effect on age at death and presence of dementia.

1 Age at death

No E2 allele mean age at death 58.5 years +3.4 n=18 E2 allele mean age at death 66.4 years +3.9 n=5

Significant difference p< 0.007 (t-test)

2 Presence of Dementia

Dementia No Yes

E2 allele No 1 17

Yes 4 1

Significant difference p< 0.002 (chi square test) These results confirm that patients with an E2 allele survive to a greater age. These results also confirm the suφrising finding that despite surviving to a greater age these patients with the E2 allele have a reduced degree of dementia.

TABLE 2

Case Age ApoE Genotype Dementia

A 52 34 yes

B 54 34 yes

C 56 34 yes

D 56 33 yes

E 57 33 yes

F 57 34 yes

G 58 33 yes

H 58 33 yes

I 58 33 yes

J 59 34 yes

K 59 34 yes

L 59 33 yes

M 59 34 yes

N 60 34 yes

P 60 33 yes

Q 61 32 no

R 64 32 no

S 65 33 yes

T 67 33 no u 68 32 yes

V 68 22 no w 71 32 no

References

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Claims

1. A method of prognosing in a subject for the course and nature of disease progression and the ultimate degree of dementia which a subject would suffer during a β amyloid-related disease, wherein the presence of an ApoE2 isoform or DNA in a subject at risk of such disease or suspected of having such a disease or in the early stages of such a disease indicates that the subject is likely to have a milder, more benign course of disease with the extent of the eventual cognitive impairment or dementia being of mild or moderate degree, the method comprising detecting the presence or absence of ApoE isoforms or of DNA encoding ApoE isoforms in the subject.

2. A method according to claim 1 wherein the subject has not previously been diagnosed as afflicted with β amyloid-related disease.

3. A method according to claim 2 wherein the subject has previously been determined to be at risk of developing β amyloid-related disease.

4. A method according to any of claims 1 to 3 wherein the subject is at risk of, suffering from or suspected of suffering from Alzheimer's disease.

5. A method according to any of claims 1 to 3wherein the subject is at risk of, suffering from or suspected of suffering from cortical Lewy body disease.

6. A method according to any of claims 1 to 3 wherein the subject is at risk of, suffering from or suspected of suffering from Parkinson's disease.

7. A method according to any of claims 1 to 3 wherein the subject is at risk of, suffering from or suspected of suffering from vascular and cerebrovascular disease which predispose to β amyloid-related diseases.

8. A method according to claim 3 wherein the subject is a DS individual.

9. A method according to any preceding claim wherein the step of detecting the presence or absence of ApoE isoforms or of DNA encoding such isoforms is carried out ex vivo.

10. A method according to claim 9 wherein said detection step involves collecting a sample of biological material containing DNA from the subject.

11. A method according to claim 10, wherein the biological sample is blood.

12. A method according to claim 9 wherein said detection step involves collecting a sample of biological material containing ApoE from the subject.

13. A method according to claim 12, wherein the biological sample is cerebrospinal fluid.

14. A method of treatment of β amyloid-related disease by the supplementation of ApoE2 protein expression.

15. A method according to claim 14 wherein said supplementation is effected by direct administration of ApoE2 isoform or by gene therapy.

16. A method according to claim 14, wherein said supplementation is effected by the generation of an agonist which mimics the action of ApoE2 on the ApoE receptors.