HK1090392A1 - Methods for the diagnosis and prognosis of alzheimer's disease - Google Patents

Abstract

The invention relates to methods for the diagnosis or prognosis of Alzheimer's disease in an individual. An inventive method has at least one step for detecting the presence or the absence of the minority allele of polymorphism rs908832 of gene ABCA2. The presence of the minority allele of polymorphism rs908832 of gene ABCA2 indicates that the individual can be afflicted with Alzheimer's disease or is presently at increased risk of developing Alzheimer's disease.

Description

Method for detecting alzheimer's disease

The present invention relates to a method for the diagnosis or prognosis of alzheimer's disease. The invention also relates to a kit for the diagnosis or prognosis of Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disease that affects a high proportion of the elderly population. Clinically, the disease is characterized by loss of cognitive function and neuropathologically by the presence in the brain of intracellular neurofibrillary deposits and extracellular deposits of beta amyloid polypeptide (a β), resulting in the formation of amyloid plaques. The starch plaques are mainly composed of a β peptides comprising 40 or 42 amino acids, which are produced by proteolysis of β Amyloid Precursor Protein (APP). Extracellular deposition of a β is specific for alzheimer's disease. They represent an early and invariant feature of all forms of alzheimer's disease, including familial forms.

The familial form of the disease occurs relatively early (between the ages of 40-60). They are due at least in part to mutations in the APP gene and presenilin-1 (PS1) and presenilin-2 (PS2) genes. Mutations in these three genes induce changes in APP proteolysis, leading to overproduction of a β, premature pathology and symptoms similar to sporadic alzheimer's disease.

The relationship between cholesterol and Alzheimer' S disease has also been established from epidemiological studies and the results of modern biochemical and cellular biological studies (see review Hartmann, T. (2001) TINS 24: S45-48). High cholesterol levels in adults as well as high arterial pressure significantly increase the risk of Alzheimer's disease (Kivipelto et al, 2001 Br Med J.322: 1447).

On the other hand, a greatly reduced risk of Alzheimer's disease was observed in people treated with statins cholesterol lowering drugs (Wolozin et al (2000) Arch neurol.57: 1439; Jick et al (2000) Lancet 356: 1627).

Recently, molecular level associations have been established. High cholesterol levels increase A β peptide production and accelerate the appearance of amyloid plaques both in vitro (in vitro) and in vivo (in vivo) (spark et al (1994), exp. neurol.126: 88-94; reflo et al (2000), neurobiol. Dis.7: 321-) -331; Puglielli et al (2001), Nat. cell biol.3: 905; Shie et al (2002), Neuroreport 13: 455), whereas inhibitors of the cholesterol synthesis pathway reduce them (Simons et al (1998), PNAS USA, 95: 6460-.

Despite significant progress, the medical community is still faced with a lack of molecules that are truly effective against alzheimer's disease. One of the reasons for this lack is the difficulty in finding targets that effectively screen for molecules that can act therapeutically against the disease.

Furthermore, early detection of the disease is crucial in order to provide treatment before the disease has not yet exhibited the most highly disabling first symptoms.

In addition, since the disease is of diverse morphology, it is clear that targeted therapy is required, firstly according to the individual to be treated and secondly according to the molecules that can be used for this therapy. Such pharmacogenomic or pharmacogenetic approaches are becoming increasingly important.

The ABCA2 protein is a protein belonging to the ABC (ATP binding cassette) class of cholesterol transporters. This transporter protein is specifically expressed in the brain.

In 2000, Zhao et al described the cloning of the ABCA2 gene (biochem. J., 350, 865-872). This sequence is also the subject of PCT application WO 01/14414, filed by the company ACTIVEPASS PHARMACEUTICALS. The application proposes various assumptions about the ABCA2 effect, but no results support these assumptions.

Different polymorphisms of the ABCA2 gene have been identified. Their sequences are available in particular from the dbSNP database of NCBI. Among the different polymorphisms listed in this pool, the rs908832 polymorphism is described.

This polymorphism is characterized in that it is the major allele of the Caucasian population with guanine at position 348 in the sequence SEQ ID No.2 and the minor allele of the Caucasian population with adenine at position 348 in the sequence SEQ ID No. 1.

Alternatively, such single nucleotide polymorphisms are represented by the sequences of SEQ ID No.3 and SEQ ID No.4 which are complementary to portions of SEQ ID No.1 and SEQ ID No.2, respectively. Thus, this polymorphism is characterized in that it is the major allele in the caucasian population with cytosine at position 201 in sequence SEQ ID No.4 and the minor allele in the caucasian population with thymidine at position 201 in sequence SEQ ID No. 3.

This polymorphism is synonymous, i.e., it does not alter the sequence of the translated protein. Both alleles of this polymorphism are part of the codon encoding aspartic acid. This coding polymorphism is located at position 2185 of exon 14 of the transcript having the sequence SEQ ID No. 5.

Surprisingly, the applicant has demonstrated that patients with the rs908832 polymorphic minor allele of the ABCA2 gene have an increased risk of premature alzheimer's disease.

This finding is particularly important because, to the best of the applicant's knowledge, it provides for the first time evidence of a relationship between the ABCA2 protein, in particular a polymorphism in the gene encoding this protein, and pathological conditions such as alzheimer's disease.

The present invention enables the confirmation and/or prognosis of the severity and possibly the desired therapeutic effect achieved in a patient already diagnosed with alzheimer's disease, as well as the risk of developing the disease in individuals whose prognosis does not show symptoms of alzheimer's disease, including individuals associated with a patient who has been diagnosed as such.

The present invention also enables to demonstrate the main functional role of ABCA2 in the pathophysiology of alzheimer's disease, which proves to be justified by the use of tests for screening molecules capable of exerting a stimulating or inhibiting effect on the activity of ABCA2 for therapeutic applications of alzheimer's disease.

Accordingly, a first object of the present invention is a method for diagnosing or prognosing an individual with alzheimer's disease. The method comprises the step of detecting at least one polymorphic allele of the ABCA2 gene. Advantageously, such polymorphisms are polymorphisms involved in alzheimer's disease. Preferably the rs908832 polymorphism, but may be any other polymorphism in genetic linkage disequilibrium with the rs908832 polymorphism.

Suitable individuals may be, for example:

-individuals not exhibiting symptoms of Alzheimer's disease,

individuals who have been detected at risk of developing Alzheimer's disease but who do not yet show symptoms of the disease, and

-individuals who have previously been diagnosed with alzheimer's disease and wish to confirm the diagnosis.

The step of detecting the presence or absence of the minor allele of the rs908832 polymorphism (which steps may or may not be performed simultaneously) may be performed directly or indirectly by any suitable method using a biological sample.

Thus, the present invention also relates to a method of screening a biological sample taken from an individual, particularly an individual who does not exhibit symptoms of Alzheimer's disease, to determine whether the individual is predisposed to developing Alzheimer's disease. The screening method comprises searching for the presence of (possibly at the same time) the minor allele of the rs908832 polymorphism in said biological sample.

The sample comprising the DNA or protein to be identified may be of various origins. For example, it may be a blood, semen, hair (with hair roots) sample or any other sample containing nucleated cells. Preferably, the biological sample analyzed is a blood sample. In this case, the DNA to be identified is derived from leukocytes.

For the purposes of the present invention, the term "biological sample" is intended to mean a sample directly from an individual for whom diagnosis or prognosis has not been made with other transformations, or a sample which has undergone one or more preparation steps and in which only components useful for the detection step, such as a crude cell extract, have been preserved.

The technique for detecting or identifying the presence or absence of DNA carrying the minor allele of the rs908832 polymorphism may be a combination of Polymerase Chain Reaction (PCR), hybridization, Southern blotting, nuclease digestion, Restriction Fragment Length Polymorphism (RFLP) and/or direct sequencing of the PCR products. All these techniques are known to the person skilled in the art.

In general, techniques for identifying DNA carrying the rs908832 polymorphic minor allele which may be used in the context of the present invention include: collection comprises The preliminary steps of The biological sample of The DNA to be identified and The step of extracting genomic DNA according to standard techniques well known to those skilled in The art, such as The method of Smith et al (The Lancet, 1992, 339, pages 1375-7).

The identification method may include:

a) extracting the DNA of the individual and extracting the DNA of the individual,

b) amplifying the isolated DNA using primers capable of amplifying the sequence corresponding to each of the rs908832 polymorphisms of the ABCA2 gene, and

c) determining at least one allele of the rs908832 polymorphism of the ABCA2 gene in the amplified DNA.

According to an advantageous embodiment, PCR techniques are used. Thus, step b) advantageously comprises a step of polymerase chain reaction. This technique involves first synthesizing oligonucleotides (primers) complementary to the sequences of the defined regions of the DNA fragment to be amplified. These oligonucleotides are used as primers for DNA polymerases. Then, the following steps are performed: heat denaturation (92-95 ℃) to separate the two DNA strands, hybridization with two specific primers by temperature reduction (50-55 ℃) and extension of the primers with polymerase DNA at 70-72 ℃.

Such primers for amplification of each allele, in particular the minor allele, in the rs908832 polymorphism of the ABCA2 gene and their complementary sequences form a further subject matter of the present application. Advantageously, they have respective sequences such that the number of bases between their respective hybridization points on the DNA molecule is between 25 and 2500 base pairs, preferably between 100 and 500 base pairs.

Advantageously, such primers have approximately 15-30 consecutive nucleotides of sequence SEQ ID No.3 or of sequence SEQ ID No. 4. Preferably, they are a pair of primers having the sequences SEQ ID No.6 and SEQ ID No. 7.

According to an advantageous embodiment, the object of the present application is a method characterized in that an amplified DNA carrying the rs908832 polymorphic minor allele is distinguished from an amplified DNA not carrying the allele by specific hybridization of two probes, each of which is specific for one of the two polymorphic forms. These probes and their complements constitute further subject matter of the present application.

Advantageously, these probes consist of approximately 12-17 consecutive nucleotides of sequence SEQ ID No.3 or sequence SEQ ID No.4, respectively. Preferably, they have the sequences of SEQ ID No.8 and SEQ ID No.9, respectively.

Amplified DNA carrying the minor allele of the rs908832 polymorphism can also be distinguished from amplified DNA not carrying the allele by techniques using the 5' nuclease activity of DNA polymerase i (taqman).

According to an advantageous embodiment, the object of the present application is a method, characterized in that an amplified DNA carrying the rs908832 polymorphism is distinguished from an amplified DNA not carrying the polymorphism by restriction fragment polymorphism (RFLP) analysis. Advantageously, the amplified DNA has been digested with restriction enzymes to give restriction fragments before migration with agarose gel, Southern blotting on a membrane and hybridization.

In order to achieve both a higher sensitivity and a better specificity, it is also possible to carry out two successive PCRs using two different pairs of primers ("nested PCR"): the first pair is an outer primer which can obtain a DNA fragment amplified as in a conventional PCR, and the second pair is an inner primer which facilitates amplification of the DNA fragment obtained by the first PCR.

In order to determine the genetic footprint of the region of interest, the DNA fragments obtained by PCR may also be separated according to their size by electrophoresis and visualized by means of EBT (ethidium bromide) and UV light.

In this case, it is particularly advantageous to be able to carry out PCR using a first primer having a mutant nucleotide sequence at the 3 'end and a second primer having a wild-type nucleotide sequence at the 3' end. The difference in denaturation temperature and hence amplification efficiency in both cases allowed the mutant DNA to be distinguished from the wild-type DNA.

Alternatively, the amplified DNA fragments are directly identified using dot blot techniques which include spotting samples of PCR-derived DNA fragments on nylon filters, denaturing the DNA fragments, hybridizing thereto with radioactive specific probes and washing to remove excess unbound radioactive products, and autoradiography. Other methods of visualization using specific probes comprising labels other than radiolabels, such as dyes or fluorescent labels, are also possible.

Alternatively, the presence of a mutation in the DNA comprising the rs908832 polymorphism may also be detected by directly sequencing all or part of the amplified DNA fragment. The method consists in determining the nucleotide sequence of the rs908832 polymorphism. Sequencing can be carried out by any method known to the person skilled in the art, for example by the Sanger method or by the Maxam and Gilbert methods.

Alternatively, the presence or absence of DNA carrying the rs908832 polymorphism may be detected using Southern blotting techniques which comprise subjecting the resulting DNA fragment to electrophoretic separation after treatment with one or more restriction enzymes. The gel was then denatured and blotted on a nylon membrane. The membrane is hybridized with a specific probe. After washing to remove excess unbound radioactive product, a film is placed over the membrane and exposed. One or more bands corresponding to the DNA fragments recognized by the probes can then be detected.

The presence or absence of the rs908832 polymorphism may also be detected using RFLP techniques in combination with Southern blotting and/or PCR techniques. RFLP can be used to compare DNA of various individuals and to investigate whether point mutations have been generated that allow the appearance or disappearance of restriction sites. Treatment of two DNAs having the same sequence with restriction enzymes will give identical fragments, and Southern blots derived from these fragments will therefore be identical. Conversely, if the restriction site disappears or appears after mutation, the fragments will no longer be of the same size, and this can be seen on autoradiographs. The same is true if new restriction sites appear after the mutation.

The rs908832 polymorphism of the ABCA2 gene may also be detected by determining the nucleotide at position 2185 of the transcript encoding ABCA2 protein.

All of the above techniques are well known to those skilled in the art. Any other technique known and also suitable for determining the presence or absence of the rs908832 polymorphism may be used. In this connection, techniques such as ligase chain reaction, strand displacement amplification, transcription dependent amplification and the like can be mentioned. Advantageously, The detection is carried out according to The Methods described in The literature (see, for example, Gugh et al, Nature, 1990, 347, page 773; Kagimoto et al, J.biol.chem., 1990, 265, page 17209; Wolf et al, The Lancet, 1990, 336, page 1452; Hayashi et al, Nucleic Acids Res., 1991, 19, page 4797; Daly et al, Pharmacogenetics, 1991, page 33; Spurr et al, Methods enzymol, 1991, 206, page 149; Armstrong et al, The Lancet, 1992, 339, page 1017; Kurth et al, am.J.of Med.Genet., 1993, page 48, page 166; Cann et al, J.neurol.Sci., 1997, page 153, page 50, Southern et al, page 141, Southern et al, PCR, procedures described in general, PCR, in general, in pages 18, or in combination with The procedures described by The references cited in U.A, SEQ ID.

All of these assays are particularly useful because they form the basis for determining whether an individual is likely to suffer from, or exhibits an increased risk of developing, Alzheimer's disease.

Accordingly, another object of the present invention relates to a method for analyzing a biological sample from an individual, the method comprising:

a) determining the genotype of the individual ABCA2 gene,

b) transforming the data obtained in a) in order to prognose the risk of developing Alzheimer's disease and the predicted therapeutic effect of the disease in said individual.

According to another aspect, the invention also relates to a set of reagents or kits for the diagnosis and prognosis of alzheimer's disease in an individual.

Such kits may be in a packet form to hold a variety of containers such as vials or tubes. Wherein each container comprises a plurality of components required to detect the presence or absence of DNA carrying the rs908832 polymorphism.

The components capable of carrying out the detection reaction are selected from those described previously. They are, for example:

a pair of primers hybridizing to a defined region of the ABCA2 gene, and optionally means necessary for carrying out an amplification reaction, or

-an oligonucleotide probe, optionally immobilized on a support and comprising a detectable label, and optionally reagents necessary for carrying out a hybridization reaction.

Another object of the invention is a method of treating alzheimer's disease comprising:

-at least one step of determining the presence or absence of the rs908832 polymorphic minor allele in the individual, and

-administering a compound or a mixture of compounds known to be active on alzheimer's disease to an individual exhibiting the rs908832 polymorphic minor allele.

Preferably, the disease is early stage alzheimer's disease.

Another object of the invention is a method of selecting a compound intended to be administered to an individual exhibiting a disease associated with the rs908832 polymorphism of the ABCA2 gene, which comprises:

a. at least one step of detecting the presence or absence of a secondary allele of the rs908832 polymorphism of the ABCA2 gene in a biological sample of said individual, and

b. selecting an appropriate compound if said allele is present.

Another object of the invention is a method for selecting a compound intended to be administered to an individual exhibiting alzheimer's disease, comprising:

a. at least one step of detecting the presence or absence of a secondary allele of the rs908832 polymorphism of the ABCA2 gene in a biological sample of said individual, and

b. selecting an appropriate compound if said allele is present.

The application also relates to the use of a compound or mixture of compounds thereof known to be active against alzheimer's disease for the manufacture of a medicament for the treatment of an individual suffering from alzheimer's disease for which the presence of the rs908832 polymorphic minor allele has been detected before treatment of the individual.

Such compounds known to be active against alzheimer's disease are for example acetylcholinesterase inhibitors (AchEI, such as donepezil (alice), galantamine (galantamine) or rivastigmine (rivastigmine)), NMDA receptor channel antagonists (such as memantine (memantine)), inhibitors of amyloid polypeptide production such as BMS 299897 or modulators of ABCA2 activity.

The compounds of these combinations may be administered orally, parenterally, transdermally or rectally, simultaneously or separately, or in a dispersed manner over a period of time.

These compounds may be formulated in the form of a pharmaceutical composition comprising one or more of the compounds defined above in combination with a pharmaceutically acceptable vehicle.

Solid compositions for oral administration may be tablets, pills, powders (gelatin capsules, cachets) or granules. For these compositions, the active ingredients are mixed under a flow of argon with one or more inert diluents, such as starch, cellulose, sugar, lactose or silica. These compositions may also contain substances other than diluents, for example one or more lubricants such as magnesium stearate or talc, colorants, sugar coatings (dragees) or paints.

Liquid compositions for oral administration may be pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable or paraffin oils. These compositions may contain substances other than diluents, such as wetting agents, sweeteners, thickeners, flavoring agents or stabilizers.

Sterile compositions for parenteral administration are preferably aqueous or non-aqueous solutions, suspensions or emulsions. As solvents or vehicles there may be mentioned water, propylene glycol, polyethylene glycol, vegetable oils, in particular olive oil, injectable organic esters such as ethyl oleate or other suitable organic solvents. These compositions may also contain adjuvants, in particular wetting agents, isotonizing agents, emulsifying agents, dispersing agents and stabilizers. Sterilization can be accomplished in a variety of ways, such as by filtration, incorporating sterilizing agents into the composition, irradiation, or heating. They may also be prepared in the form of sterile solid compositions which may be dissolved in sterile water or any other injectable sterile medium at the time of use.

Compositions for rectal administration are suppositories or rectal capsules which contain, in addition to the active product, excipients such as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

Pharmaceutical compositions containing a combination as defined above will generally contain from 0.1 to 500mg of the compound.

The dosage depends on the desired effect, the duration of the treatment and the route of administration used; in general, the dosage will be 0.1-500mg of the compound per day for oral administration to an adult.

Generally, the physician will determine the appropriate dosage depending on the age, weight and all other individual-specific factors of the individual to be treated.

The invention also relates to a transgenic animal in which at least one foreign DNA sequence carrying the rs908832 polymorphic minor allele is inserted into its genome such that the function of the ABCA2 gene is modified.

The term "transgenic animal" is intended to refer to any non-human animal having an artificially modified genome. The genomic modification may be the result of one or more gene alterations or modifications by "knock-in" or "knock-out" (inactivation/modification of genes by homologous recombination), or the result of overexpression of a human gene in a mouse under the control of a neuronal cell type specific promoter (e.g., ThyI, PDGF or prion) or a glial cell type specific promoter (e.g., Glial Fibrillary Acidic Protein (GFAP)). This modification may be the result of conventional alteration or mutagenic action, or by stable insertion of an expression cassette capable of expressing the hybrid gene. In particular, it can be carried out, for example, according to the same or similar methods as described in application WO 01/02552 or WO 01/13176.

Modification of the genome may also be achieved by insertion or substitution of one or more genes in their wild-type or mutant form.

Advantageously, the reproductive stem cells are modified.

Since only stem cells (the term "ES" cells) are available that have the ability to colonize the germ line of mouse embryos, current genomic modifications using "knock-in" or "knock-out" techniques are limited to mice as models. While ES cell lines are available in other species, those skilled in the art will readily apply these techniques to other species to generate knock-out (KO) and/or knock-in (KI) models. Furthermore, methods based on oligonucleotides (DNA, RNA or hybrids) alone or in combination with DNA/RNA modifying enzymes can be used to introduce defined modifications/mutations at specific locations in the genome. Even radiation and chemical mutagens can be used if they induce random modifications in the genome can be combined with a useful set of biomarkers (phenotypes) and high-throughput positional cloning methods.

However, the most straightforward way to modify the genome of a laboratory animal (mouse, rat, cow, pig, sheep, etc.) is to randomly integrate the transgene by microinjecting linearized DNA into one or both pronuclei of the fertilized oocyte at a single cell stage (preferably to avoid the production of chimeric animals, although injection can also be performed at two or more cell stages). Conventionally, a transgene is composed of two parts: regulatory elements controlling the temporal and spatial expression of the RNA encoded by the DNA and said juxtaposed DNA (cDNA or genomic fragments). The two elements (regulatory element and DNA encoding the protein of interest) may be homologous or heterologous to the target genome. The transgenic animal of interest is typically selected from non-human mammals. For example, it may be a mouse (i.e., mouse, rat, and guinea pig), rabbit, cat, dog, sheep, or cow. Preferably, they are mice, rats or rabbits obtained according to conventional transgenic techniques. Another object of the invention are stem cell lines and cell lines differentiated from these, in the genome of which at least one exogenous genomic DNA sequence carrying the minor allele of the rs908832 polymorphism has been inserted.

Briefly, the transgenic animals, stem cell lines and differentiated cell lines of the invention were generated by inserting exogenous genomic DNA encoding the ABCA2 protein into the corresponding gene of the animal using homologous recombination (the transgene is inserted immediately after the promoter of the animal gene in order to force the transgene into the correct expression pattern and prevent the animal's endogenous gene expression: knock-out), or by inserting a specific mutation described in the present invention corresponding to another isoform of the human ABCA2 gene into the animal's endogenous gene (modified by homologous recombination: knock-in stem cells), or by generating a transgenic animal by overexpression of the ABCA2 gene carrying the minor allele of the rs908832 polymorphism.

In the case of mice, these animals can advantageously be crossed with transgenic mice carrying the human APP gene with the Alzheimer's type mutation and developing into starch plaques. The double transgenic animals thus obtained reproduce the genotype observed in patients suffering from or at risk of Alzheimer's disease. The resulting neonatal animal may then be genotyped for the rs908832 polymorphism using techniques already described above, in particular using amplification reactions (PCR) and/or Southern blotting.

Such transgenic animals are particularly valuable because they provide an advantageous model for understanding alzheimer's disease, which model reproduces the characteristics of alzheimer's disease very realistically. In contrast to known models, this model can be used in particular to demonstrate compounds which are particularly suitable for the treatment of alzheimer's disease, in particular said human alzheimer's disease. These compounds may be chemical molecules, peptide or protein molecules, antibodies, chimeric molecules, and antisense DNA or ribozymes.

The compounds identified can be used as pharmaceutical products, either as such or in combination with a pharmaceutically acceptable vehicle, in order to obtain a pharmaceutical composition. They are, in particular, sterile isotonic saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts), or dry compositions, in particular lyophilized compositions, in which case injectable solutions may be formulated when appropriate amounts of sterile water or physiological saline are added. Can be injected by three-dimensional stereotaxic, topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, and transdermal methods.

The confirmation of the aforementioned compounds is based on: the animal models of the invention are contacted with a compound or mixture of compounds that is supposed to have an effect, e.g. by injection administration, and the effect of the compounds on various biochemical and/or histological changes, in particular in the brain of the model, is determined.

These transgenic animals can be used for in vivo testing of therapeutic compounds for prevention, attenuation or cure of Alzheimer's disease, and can also provide Alzheimer's disease animal models for screening environmental factors for induction or acceleration of pathogenesis, studying behavior during disease development, and studying various biological mechanisms involved therein, for the purpose of studying new medical products or determining the effective amount and toxicity of medical products. Therefore, another object of the present invention relates to the use of a transgenic animal, stem cell line or differentiated cell line as described previously for testing the activity of a compound or method intended for the prevention and/or treatment of alzheimer's disease. Another object of the invention relates to cells extracted from transgenic animals such as those described previously, and to the use thereof in compounds demonstrated to be intended for the treatment of alzheimer's disease.

The above compounds were confirmed to be based on: contacting cells extracted from the animal model of the invention with a compound or mixture of compounds that is supposed to have an effect, and determining the effect of the compounds on various parameters of whole cells, cell homogenates or subcellular components, such as cell death.

In addition to those listed above, the present invention also comprises other features and advantages deriving from the following examples and figures, which should be considered as illustrating the invention and not limiting its scope.

Examples

Example 1: investigating the genotype of the rs908832 polymorphism of the ABCA2 gene in a patient sample

After consent, DNA from patients of Caucasian descent with Alzheimer's disease and control individuals was used.

The frequency of polymorphisms was determined by determining the genotype of a 47 individual sample sold by the Coriell institute (USA).

To determine the genotypes of patients and controls with alzheimer's disease, ten polymorphisms (including the rs908832 polymorphism) were selected based on their location in the gene and their frequency.

Genotyping was performed by 5 'nuclease assay (allele-discriminating technique from Applied Biosystems, Foster City, usa) and then statistical analysis of Chi-square test (Chi2) was performed by testing the association of each of these markers in alzheimer's disease. For analysis, the disease was grouped by age of onset (early onset (pre-65 or 65) or late onset (post-65)) and its source (sporadic (first known case) or familial (other cases already in the same family)).

The results obtained for the rs908832 polymorphism are as follows:

-frequency of minor alleles (T) in the patient (440 genotypes): 7.4

Frequency of minor alleles (T) in control (519 genotypes): 3.4.

the hardy-weinberg ratios of patients and controls were confirmed: no significant bias was observed, confirming the absence of genotype errors.

Results of the development test in patients presenting with early forms of disease (pre-65 years or 65 years):

136 sporadic cases versus 272 controls:

-heterogeneity assay: chi2(1ddl) ═ 22.69p ═ 2 × 10^-6

-allele test (frequency (T) vs frequency (C)): chi2(1ddl) ═ 21.27p ═ 4 × 10^-6

104 familial cases versus 272 controls:

-heterogeneity assay: chi2(2ddl) ═ 7.80p ═ 0.02

-allele test (frequency (T) vs frequency (C)): chi2(1ddl) ═ 7.27p ═ 7 × 10^-3。

Approximate odd ratio (adjustment for gender and APOE-e 4 status) was estimated by logistic regression on 240 patients presenting early morphology. The results are also significant: OR 3.97IC ═ 2.23-7.09.

Even after multiple tests of the correlation by the Bonferroni method, the values obtained were still significant (threshold fixed at 0.05).

These results indicate that the ABCA2 gene is an important gene in Alzheimer's disease etiology.

This result is of the same order of magnitude as the risk conferred by the apolipoprotein E4, which apolipoprotein E4 has hitherto been known to be the highest risk factor for alzheimer's disease.

Example 2: generation of transgenic mice

1. Generation of transgenic mice expressing human ABCA2 protein

By using e.g. Sculptor^TM(Amersham, France) in vitro mutagenesis System mutagenesis of human ABCA2 protein was performed. The coding region of ABCA2 was subcloned into a Bluescript type cloning vector (stratagene) and mutations were introduced according to the manufacturer's protocol using oligonucleotides containing the mutations of interest. The mutated sequence was confirmed by sequence analysis.

Generation and characterization of ABCA2 transgenic mice

To construct a transgene, genomic DNA encoding ABCA2 and exhibiting the human rs908832 polymorphic minor allele is subcloned into a multi-site junction of a vector for specific expression of the transgene in certain tissue/cell types, such as THYI (Luthi et al, J.Neuroscience, 17, 4688-99) specific for neuronal cell types, PDGF or prions or GFAP specific for glial cell types. Supercoiled DNA was prepared using a plasmid preparation kit (Qiagen). For microinjection, the vector sequences must be removed using defined restriction enzyme digestion, leaving the transgene intact and isolated from unwanted cloning vector sequences. The fragment containing the expression cassette was then purified by agarose gel electrophoresis.

The microinjected aliquots were dialyzed against TE buffer (10mM Tris pH 7.4; 0.1mM EDTA) on a floating filter (Millipore; membrane type: VS; 0.025 μm) and then filtered (Spin-X; Costar; polyvinyl acetate; 0.22 μm). For microinjection, the DNA was diluted to a final concentration of 1-2 ng/. mu.l. The purified fragment was injected into one of the two pronuclei of mouse fertilized oocytes. The surviving embryos were immediately transplanted into the oviducts of the female mice (pseudopregnancy). The presence of the transgene in the neonatal mouse is determined either by PCR or by Southern analysis using specific probes/sequences. With all these analyses, any major rearrangements or deletions of transgenes in founders and their progeny can be excluded.

3. Production of transgenic animals containing human ABCA2 protein in their genome

The ABCA2 gene having the minor allele of the human rs908832 polymorphism was introduced into a predetermined target position of the mouse gene using stem cell homologous recombination techniques. Homozygous mouse genomic libraries (lambda, BAC, YAC, etc. libraries) can be easily screened using the primers and samples described herein to isolate and clone the corresponding mouse genes. The genomic organization of the mouse gene and its sequence can be characterized using standard techniques known to those skilled in the art (restriction site mapping, sequencing, bioanalytical tools) to determine the exact location where human DNA should be inserted to obtain the desired expression pattern of human DNA and ultimately interrupt the expression of the mouse gene. Once the exact location is identified, standard targeting vectors for stem cells (i.e., vectors with markers for selecting for the intended insertion event, all of which are well known to those skilled in the art) can be constructed. Rapidly, the selection cassette (gene for resistance to antibiotics) was placed within the confines of the 3 'and 5' ends of the mouse genomic DNA fragment (2-6kb), which equates to the 3 'and 5' extensions of the sequence of the murine ABCA2 gene immediately following insertion into the selected site.

Based on the knowledge of the murine gene, in order to optimize and work with the high throughput screening method, a positive control vector was generated that screens for recombinant stem cells (once successfully integrated, the vector replicated the locus of the murine gene).

DNA was purified for targeting experiments according to standard methods. The targeting vector is introduced into the stem cells using standardized electroporation techniques, after which the cell clones are subjected to a continuous screening process (antibiotics) to promote the growth of stem cell clones carrying the recombinant of interest. Generally, screening is performed for about 2 weeks. Resistant stem cell clones were screened by PCR and/or Southern.

Stem cells with the desired recombination without any other detectable modification in their genome are clonally grown to obtain sufficient cells. The cells were then injected into day 3 semi-embryos obtained from naturally ovulating mothers. Surviving embryonic cells (containing stem cells) are implanted into the recipient mother where they develop to completion and give rise to neonatal mice consisting of cells derived from host embryonic cells and stem cell clones. This class of animals is called "chimeric animals".

These chimeras (preferably male, since most stem cell lines are derived from male mice) are "mated" with wild type mice to obtain animals in which the modification is heterozygous. The heterozygote animals were bred to produce homozygote animals.

4. Production of transgenic animals containing the minor allele of the rs908832 polymorphism in the human ABCA2 gene in its corresponding gene

In the same manner as described above, the murine gene was isolated and characterized. For such modifications, it is necessary to compare the human and murine genes in order to identify the exact location in the murine gene into which a point mutation in the human gene must be introduced. At this stage, biological analysis is necessary. Finally, in the same manner as described above, a targeting vector is developed and assembled. After successful homologous recombination, only the point mutation of interest is present in the coding sequence of the murine gene. Some selectable markers may remain in the intron, but they generally have no effect on gene expression. If necessary, they can be removed using second generation targeting vectors containing recombinase recognition elements. Once the construct has been assembled, the subsequent steps are the same as the method steps described previously.

Typically, to promote deposition of starch plaques, these ABCA2 transgenic mice were crossed with transgenic mice expressing the APP gene carrying the alzheimer's familial form mutation.

5. Neural histopathology

Brain tissue preparation

Mice were deeply anesthetized (pentobarbital i.p. 60mg/ml/kg, ketamine i.p. 40mg/ml/kg) and then perfused first with normal saline and then with paraformaldehyde (4% in PBS) via the heart. The brains were then removed and postfixed with the same fixative for 24 hours at 4 ℃. After fixation, the brains were divided into left and right half brains and paraffin embedded using standard methods.

The left half-brain of transgenic and non-transgenic mice was paraffin-embedded, and post-mortem human brain tissue (prefrontal cortex) blocks from patients with alzheimer's disease and controls were also paraffin-embedded and cut into 6 μm thick sections (serial sections) using a microtome (LEICA RM 2155, france). Tissue pieces of right half brains of transgenic and non-transgenic mice were cut to a thickness of 25 μm.

The immunoreactivity of amyloid polypeptide a β was examined as generally described by those skilled in the art.

Sequence listing

<110> Anduwan Special medicine stocks Co., Ltd

<120> method for detecting Alzheimer's disease

<130>PRJ03027

<160>9

<170> PatentIn version 3.1

<210>1

<211>562

<212>DNA

<213> human (Homo sapiens)

<400>1

ttcatcacct gcgggtgggc caggggcttg gggcaggccc cggggaggac gccgcccctc 60

cctgccagcc cgcgcctcca gggagagtcc cggcccgcgc acctccttga gccggtgctc 120

cttctccgcc acgatgtgct ggatggtcat ggccacggag tagacccagg agatcaccat 180

gcacagcggc atcatgtgct caatgacaaa caggaagctg cggggaggcc gcgctcaggc 240

gccactcagc cccagcccca gccccagccc cgggcgccca gcactcactc atcgcgtgtg 300

tagcaggggt aggggaacat ctgcacgtag ctgcctggct ccaccacatc gtgccccaca 360

aaagtgtcga tgatggcgcg ctccatcatg tctgtgggtg ggggcagcca tcaggtgccg 420

ggcaggccct ctcgtcctca cacctgtcct cccccatgaa tcctccagcc ggtcttccgg 480

gcctgctcct caccctggat ccagacgaag ccgtagagga agtagaagcg gccgccagta 540

ttgggcccag gccgccagta gg 562

<210>2

<211>562

<212>DNA

<213> human

<400>2

ttcatcacct gcgggtgggc caggggcttg gggcaggccc cggggaggac gccgcccctc 60

cctgccagcc cgcgcctcca gggagagtcc cggcccgcgc acctccttga gccggtgctc 120

cttctccgcc acgatgtgct ggatggtcat ggccacggag tagacccagg agatcaccat 180

gcacagcggc atcatgtgct caatgacaaa caggaagctg cggggaggcc gcgctcaggc 240

gccactcagc cccagcccca gccccagccc cgggcgccca gcactcactc atcgcgtgtg 300

tagcaggggt aggggaacat ctgcacgtag ctgcctggct ccaccacgtc gtgccccaca 360

aaagtgtcga tgatggcgcg ctccatcatg tctgtgggtg ggggcagcca tcaggtgccg 420

ggcaggccct ctcgtcctca cacctgtcct cccccatgaa tcctccagcc ggtcttccgg 480

gcctgctcct caccctggat ccagacgaag ccgtagagga agtagaagcg gccgccagta 540

ttgggcccag gccgccagta gg 562

<210>3

<211>400

<212>DNA

<213> human

<400>3

gggcccaata ctggcggccg cttctacttc ctctacggct tcgtctggat ccagggtgag 60

gagcaggccc ggaagaccgg ctggaggatt catgggggag gacaggtgtg aggacgagag 120

ggcctgcccg gcacctgatg gctgccccca cccacagaca tgatggagcg cgccatcatc 180

gacacttttg tggggcacga tgtggtggag ccaggcagct acgtgcagat gttcccctac 240

ccctgctaca cacgcgatga gtgagtgctg ggcgcccggg gctggggctg gggctggggc 300

tgagtggcgc ctgagcgcgg cctccccgca gcttcctgtt tgtcattgag cacatgatgc 360

cgctgtgcat ggtgatctcc tgggtctact ccgtggccat 400

<210>4

<211>400

<212>DNA

<213> human

<400>4

gggcccaata ctggcggccg cttctacttc ctctacggct tcgtctggat ccagggtgag 60

gagcaggccc ggaagaccgg ctggaggatt catgggggag gacaggtgtg aggacgagag 120

ggcctgcccg gcacctgatg gctgccccca cccacagaca tgatggagcg cgccatcatc 180

gacacttttg tggggcacga cgtggtggag ccaggcagct acgtgcagat gttcccctac 240

ccctgctaca cacgcgatga gtgagtgctg ggcgcccggg gctggggctg gggctggggc 300

tgagtggcgc ctgagcgcgg cctccccgca gcttcctgtt tgtcattgag cacatgatgc 360

cgctgtgcat ggtgatctcc tgggtctact ccgtggccat 400

<210>5

<211>8154

<212>DNA

<213> human

<400>5

ggggcggagc cagcgcggat cgggtcccgg acgcccgagc gccccgcccc cgcgcgggcg 60

atgcccagcg gcgcggcggg ctgcggggcc cggcggggcg cgcagaggag cgggccgcgg 120

cgctgaggcg gcggagcgtg gccccgccat gggcttcctg caccagctgc agctgctgct 180

ctggaagaac gtgacgctca aacgccggag cccgtgggtc ctggccttcg agatcttcat 240

ccccctggtg ctgttcttta tcctgctggg gctgcgacag aagaagccca ccatctccgt 300

gaaggaagtc cccttctaca cagcggcgcc cctgacgtct gccggcatcc tgcctgtcat 360

gcaatcgctg tgcccggacg gccagcgaga cgagttcggc ttcctgcagt acgccaactc 420

cacggtcacg cagctgcttg agcgcctgga ccgcgtggtg gaggaaggca acctgtttga 480

cccagcgcgg cccagcctgg gctcagagct cgaggcccta cgccagcatc tggaggccct 540

cagtgcgggc ccgggcacct cggggagcca cctggacaga tccacagtgt cttccttctc 600

tctggactcg gtggccagaa acccgcagga gctctggcgt ttcctgacgc aaaacttgtc 660

gctgcccaat agcacggccc aagcactctt ggccgcccgt gtggacccgc ccgaggtcta 720

ccacctgctc tttggtccct catctgccct ggattcacag tctggcctcc acaagggtca 780

ggagccctgg agccgcctag ggggcaatcc cctgttccgg atggaggagc tgctgctggc 840

tcctgccctc ctggagcagc tcacctgcac gccgggctcg ggggagctgg gccggatcct 900

cactgtgcct gagagtcaga agggagccct gcagggctac cgggatgctg tctgcagtgg 960

gcaggctgct gcgcgtgcca ggcgcttctc tgggctgtct gctgagctcc ggaaccagct 1020

ggacgtggcc aaggtctccc agcagctggg cctggatgcc cccaacggct cggactcctc 1080

gccacaggcg ccacccccac ggaggctgca ggcgcttctg ggggacctgc tggatgccca 1140

gaaggttctg caggatgtgg atgtcctgtc ggccctggcc ctgctactgc cccagggtgc 1200

ctgcactggc cggacccccg gacccccagc cagtggtgcg ggtggggcgg ccaatggcac 1260

tggggcaggg gcagtcatgg gccccaacgc caccgctgag gagggcgcac cctctgctgc 1320

agcactggcc accccggaca cgctgcaggg ccagtgctca gccttcgtac agctctgggc 1380

cggcctgcag cccatcttgt gtggcaacaa ccgcaccatt gaacccgagg cgctgcggcg 1440

gggcaacatg agctccctgg gcttcacgag caaggagcag cggaacctgg gcctcctcgt 1500

gcacctcatg accagcaacc ccaaaatcct gtacgcgcct gcgggctctg aggtcgaccg 1560

cgtcatcctc aaggccaacg agacttttgc ttttgtgggc aacgtgactc actatgccca 1620

ggtctggctc aacatctcgg cggagatccg cagcttcctg gagcagggca ggctgcagca 1680

acacctgcgc tggctgcagc agtatgtagc agagctgcgg ctgcaccccg aggcactgaa 1740

cctgtcactg gatgagctgc cgccggccct gagacaggac aacttctcgc tgcccagtgg 1800

catggccctc ctgcagcagc tggataccat tgacaacgcg gcctgcggct ggatccagtt 1860

catgtccaag gtgagcgtgg acatcttcaa gggcttcccc gacgaggaga gcattgtcaa 1920

ctacaccctc aaccaggcct accaggacaa cgtcactgtt tttgccagtg tgatcttcca 1980

gacccggaag gacggctcgc tcccgcctca cgtgcactac aagatccgcc agaactccag 2040

cttcaccgag aaaaccaacg agatccgccg cgcctactgg cggcctgggc ccaatactgg 2100

cggccgcttc tacttcctct acggcttcgt ctggatccag gacatgatgg agcgcgccat 2160

catcgacact tttgtggggc acgacgtggt ggagccaggc agctacgtgc agatgttccc 2220

ctacccctgc tacacacgcg atgacttcct gtttgtcatt gagcacatga tgccgctgtg 2280

catggtgatc tcctgggtct actccgtggc catgaccatc cagcacatcg tggcggagaa 2340

ggagcaccgg ctcaaggagg tgatgaagac catgggcctg aacaacgcgg tgcactgggt 2400

ggcctggttc atcaccggct ttgtgcagct gtccatctcc gtgacagcac tcaccgccat 2460

cctgaagtac ggccaggtgc ttatgcacag ccacgtggtc atcatctggc tcttcctggc 2520

agtctacgcg gtggccacca tcatgttctg cttcctggtg tctgtgctgt actccaaggc 2580

caagctggcc tcggcctgcg gtggcatcat ctacttcctg agctacgtgc cctacatgta 2640

cgtggcgatc cgagaggagg tggcgcatga taagatcacg gccttcgaga agtgcatcgc 2700

gtccctcatg tccacgacgg cctttggtct gggctctaag tacttcgcgc tgtatgaggt 2760

ggccggcgtg ggcatccagt ggcacacctt cagccagtcc ccggtggagg gggacgactt 2820

caacttgctc ctggctgtca ccatgctgat ggtggacgcc gtggtctatg gcatcctcac 2880

gtggtacatt gaggctgtgc acccaggcat gtacgggctg ccccggccct ggtacttccc 2940

actgcagaag tcctactggc tgggcagtgg gcggacagaa gcctgggagt ggagctggcc 3000

gtgggcacgc accccccgcc tcagtgtcat ggaggaggac caggcctgtg ccatggagag 3060

ccggcgcttt gaggagaccc gtggcatgga ggaggagccc acccacctgc ctctggttgt 3120

ctgcgtggac aaactcacca aggtctacaa ggacgacaag aagctggccc tgaacaagct 3180

gagcctgaac ctctacgaga accaggtggt ctccttcttg ggccacaacg gggcgggcaa 3240

gaccaccacc atgtccatcc tgaccggcct gttccctcca acgtcgggtt ccgccaccat 3300

ctacgggcac gacatccgca cggagatgga tgagatccgc aagaacctgg gcatgtgccc 3360

gcagcacaat gtgctctttg accggctcac ggtggaggaa cacctctggt tctactcacg 3420

gctcaagagc atggctcagg aggagatccg cagagagatg gacaagatga tcgaggacct 3480

ggagctctcc aacaaacggc actcactggt gcagacattg tcgggtggca tgaagcgcaa 3540

gctgtccgtg gccatcgcct tcgtgggcgg ctctcgcgcc atcatcctgg acgagcccac 3600

ggcgggcgtg gacccctacg cgcgccgcgc catctgggac ctcatcctga agtacaagcc 3660

aggccgcacc atccttctgt ccacccacca catggatgag gctgacctgc ttggggaccg 3720

cattgccatc atctcccatg ggaagctcaa gtgctgcggc tccccgctct tcctcaaggg 3780

cacctatggc gacgggtacc gcctcacgct ggtcaagcgg cccgccgagc cggggggccc 3840

ccaagagcca gggctggcat ccagcccccc aggtcgggcc ccgctgagca gctgctccga 3900

gctccaggtg tcccagttca tccgcaagca tgtggcctcc tgcctgctgg tctcagacac 3960

aagcacggag ctctcctaca tcctgcccag cgaggccgcc aagaaggggg ctttcgagcg 4020

cctcttccag cacctggagc gcagcctgga tgcactgcac ctcagcagct tcgggctgat 4080

ggacacgacc ctggaggaag tgttcctcaa ggtgtcggag gaggatcagt cgctggagaa 4140

cagtgaggcc gatgtgaagg agtccaggaa ggatgtgctc cctggggcgg agggcccggc 4200

gtctggggag ggtcacgctg gcaatctggc ccggtgctcg gagctgaccc agtcgcaggc 4260

atcgctgcag tcggcgtcat ctgtgggctc tgcccgtggc gacgagggag ctggctacac 4320

cgacgtctat ggcgactacc gccccctctt tgataaccca caggacccag acaatgtcag 4380

cctgcaagag gtggaggcag aggccctgtc gagggtcggc cagggcagcc gcaagctgga 4440

cggcgggtgg ctgaaggtgc gccagttcca cgggctgctg gtcaaacgct tccactgcgc 4500

ccgccgcaac tccaaggcac tcttctccca gatcttgctg ccagccttct tcgtctgcgt 4560

ggccatgacc gtggccctgt ccgtcccgga gattggtgat ctgcccccgc tggtcctgtc 4620

accttcccag taccacaact acacccagcc ccgtggcaat ttcatcccct acgccaacga 4680

ggagcgccgc gagtaccggc tgcggctatc gcccgacgcc agcccccagc agctcgtgag 4740

cacgttccgg ctgccgtcgg gggtgggtgc cacctgcgtg ctcaagtctc ccgccaacgg 4800

ctcgctgggg cccacgttga acctgagcag cggggagtcg cgcctgctgg cggctcggtt 4860

cttcgacagc atgtgtctgg agtccttcac acaggggctg ccactgtcca atttcgtgcc 4920

acccccaccc tcgcccgccc catctgactc gccagcgtcc ccggatgagg acctgcaggc 4980

ctggaacgtc tccctgccgc ccaccgctgg gccagaaatg tggacgtcgg caccctccct 5040

gccgcgcctg gtacgggagc ccgtccgctg cacctgctct gcgcagggca ccggcttctc 5100

ctgccccagc agtgtgggcg ggcacccgcc ccagatgcgg gtggtcacag gcgacatcct 5160

gaccgacatc accggccaca atgtctctga gtacctgctc ttcacctccg accgcttccg 5220

actgcaccgg tatggggcca tcacctttgg aaacgtcctg aagtccatcc cagcctcatt 5280

tggcaccagg gccccaccca tggtgcggaa gatcgcggtg cgcagggctg cccaggtttt 5340

ctacaacaac aagggctatc acagcatgcc cacctacctc aacagcctca acaacgccat 5400

cctgcgtgcc aacctgccca agagcaaggg caacccggcg gcttacggca tcaccgtcac 5460

caaccacccc atgaataaga ccagcgccag cctctccctg gattacctgc tgcagggcac 5520

ggatgtcgtc atcgccatct tcatcatcgt ggccatgtcc ttcgtgccgg ccagcttcgt 5580

tgtcttcctc gtggccgaga agtccaccaa ggccaagcat ctgcagtttg tcagcggctg 5640

caaccccatc atctactggc tggcgaacta cgtgtgggac atgctcaact acctggtccc 5700

cgctacctgc tgtgtcatca tcctgtttgt gttcgacctg ccggcctaca cgtcgcccac 5760

caacttccct gccgtcctct ccctcttcct gctctatggg tggtccatca cgcccatcat 5820

gtacccggcc tccttctggt tcgaggtccc cagctccgcc tacgtgttcc tcattgtcat 5880

caatctcttc atcggcatca ccgccaccgt ggccaccttc ctgctacagc tcttcgagca 5940

cgacaaggac ctgaaggttg tcaacagtta cctgaaaagc tgcttcctca ttttccccaa 6000

ctacaacctg ggccacgggc tcatggagat ggcctacaac gagtacatca acgagtacta 6060

cgccaagatt ggccagtttg acaagatgaa gtccccgttc gagtgggaca ttgtcacccg 6120

cggactggtg gccatggcgg ttgagggcgt cgtgggcttc ctcctgacca tcatgtgcca 6180

gtacaacttc ctgcggcggc cacagcgcat gcctgtgtct accaagcctg tggaggatga 6240

tgtggacgtg gccagtgagc ggcagcgagt gctccgggga gacgccgaca atgacatggt 6300

caagattgag aacctgacca aggtctacaa gtcccggaag attggccgta tcctggccgt 6360

tgaccgcctg tgcctgggtg tgcgtcctgg cgagtgcttc gggctcctgg gcgtcaacgg 6420

tgcgggcaag accagcacct tcaagatgct gaccggcgac gagagcacga cggggggcga 6480

ggccttcgtc aatggacaca gcgtgctgaa ggagctgctc caggtgcagc agagcctcgg 6540

ctactgcccg cagtgtgacg cgctgttcga cgagctcacg gcccgggagc acctgcagct 6600

gtacacgcgg ctgcgtggga tctcctggaa ggacgaggcc cgggtggtga agtgggctct 6660

ggagaagctg gagctgacca agtacgcaga caagccggct ggcacctaca gcggcggcaa 6720

caagcggaag ctctccacgg ccatcgccct cattgggtac ccagccttca tcttcctgga 6780

cgagcccacc acaggcatgg accccaaggc ccggcgcttc ctctggaacc tcatcctcga 6840

cctcatcaag acagggcgtt cagtggtgct gacatcacac agcatggagg agtgcgaggc 6900

gctgtgcacg cggctggcca tcatggtgaa cggtcgcctg cggtgcctgg gcagcatcca 6960

gcacctgaag aaccggtttg gagatggcta catgatcacg gtgcggacca agagcagcca 7020

gagtgtgaag gacgtggtgc ggttcttcaa ccgcaacttc ccggaagcca tgctcaagga 7080

gcggcaccac acaaaggtgc agtaccagct caagtcggag cacatctcgc tggcccaggt 7140

gttcagcaag atggagcagg tgtctggcgt gctgggcatc gaggactact cggtcagcca 7200

gaccacactg gacaatgtgt tcgtgaactt tgccaagaag cagagtgaca acctggagca 7260

gcaggagacg gagccgccat ccgcactgca gtcccctctc ggctgcttgc tcagcctgct 7320

ccggccccgg tctgccccca cggagctccg ggcacttgtg gcagacgagc ccgaggacct 7380

ggacacggag gacgagggcc tcatcagctt cgaggaggag cgggcccagc tgtccttcaa 7440

cacggacacg ctctgctgac cacccagagc tgggccaggg aggacacgct ccactgacca 7500

cccagagctg ggccagggac tcaacaatgg ggacagaagt cccccagtgc ctgccagggc 7560

ctggagtgga ggttcaggac caaggggctt ctggtcctcc agcccctgta ctcggccatg 7620

ccctgcggtc actgcggttg ccgcccctaa ttgtgccaaa ggctgacccg gcccgggctg 7680

cgtacaccct tgccctgctt tgccttaaag cctcggggtc tgcccggccc ctcgcccctg 7740

cctggcactg ctcaccgccc aaggcgacgc cggctggacc aggcactgct ggcctttctc 7800

ctgcccggcc tcggaaccag cttttctctc ttacgatgaa ggctgatgcc gagagcgggc 7860

tgtgggcgga gctgggtcag tcccgtattt attttgcttt gagaagaggc tcctctggcc 7920

ctgctctcct gcagggaggt ggctgtcccg cgggaagcca tcagcttggg ccagctggca 7980

ggtggcagga atggagaagc tgaccctgct ggccaggcaa ggggccagac cccccccaac 8040

ccccagctgc catcgctctc ccacccagct tggccccctg cccgcccacc tccctgggag 8100

ccgggcctgt acatagcgca cagatgtttg ttttaaataa ataaacaaaa tgtc 8154

<210>6

<211>16

<212>DNA

<213> human

<400>6

agcgcgccat catcga 16

<210>7

<211>20

<212>DNA

<213> human

<400>7

cactcatcgc gtgtgtagca 20

<210>8

<211>15

<212>DNA

<213> human

<400>8

cacgatgtgg tggag 15

<210>9

<211>14

<212>DNA

<213> human

<400>9

cacgacgtgg tgga 14

Claims (23)

1. A pair of primers for amplifying the rs908832 polymorphic secondary allele, wherein one primer has 15-30 consecutive nucleotides of sequence SEQ ID No.3 and the other primer has 15-30 consecutive nucleotides of sequence complementary to SEQ ID No.3, and the secondary allele has thymidine at position 201 of sequence SEQ ID No. 3.

2.A pair of primers as claimed in claim 1, wherein the sequence of one primer is shown as SEQ ID NO.6 and the sequence of the other primer is shown as SEQ ID NO. 7.

3. A set of reagents or kits for the diagnosis and prognosis of alzheimer's disease comprising a pair of primers according to claim 1 or 2.

4. Use of a pair of primers according to claim 1 or 2 for the preparation of a set of reagents or kits for the diagnosis or prognosis of alzheimer's disease according to claim 3.

5. Use of a pair of primers as claimed in claim 1 or 2 in the preparation of a set of reagents or kits for detecting rs908832 polymorphism of the ABCA2 gene, wherein said detection comprises determination of nucleotide 2185 of a transcript encoding ABCA2 protein.

6. The use of claim 5, wherein said detecting is performed on a biological sample.

7. The use of claim 6, wherein the biological sample is a sample comprising nucleated cells.

8. Use according to claim 6 or 7, wherein the biological sample is a blood sample, a semen sample or a hair sample.

9. The use according to claim 5, wherein the detection of the presence or absence of DNA carrying a polymorphism is performed by Polymerase Chain Reaction (PCR), hybridization, Southern blotting on a membrane, nuclease digestion, Restriction Fragment Length Polymorphism (RFLP), or direct sequencing techniques or a combination thereof.

10. The use of claim 9, wherein the detecting comprises:

a. the DNA of an individual is extracted,

b. amplifying the isolated DNA using primers capable of amplifying a sequence corresponding to the rs908832 polymorphism of the ABCA2 gene,

c. determining the presence of at least one polymorphic allele of the rs908832 of the ABCA2 gene in the amplified DNA.

11. The use of claim 10, wherein the amplification step b comprises Polymerase Chain Reaction (PCR).

12. The use of claim 10, wherein the amplified DNA carrying the rs908832 polymorphic minor allele is distinguished from the amplified DNA not carrying the allele by a technique using the 5' nuclease activity of DNA polymerase i (taqman).

13. The use as claimed in claim 10, wherein the amplified DNA carrying the rs908832 polymorphic minor allele is distinguished from the amplified DNA not carrying the allele by Restriction Fragment Length Polymorphism (RFLP) analysis.

14. Use according to claim 13, wherein the restriction fragments have been obtained by digestion of the amplified DNA with restriction enzymes before migration with agarose gel, Southern blotting and hybridization on membranes.

15. Use of a pair of primers of claim 1 in the preparation of a set of reagents or kits for analyzing a biological sample from an individual, wherein the analysis comprises:

a. determining the genotype of the ABAC2 gene, and

b. converting the data obtained so as to prognose the risk of developing Alzheimer's disease and the predicted therapeutic effect of the disease in said individual.

16. The use of claim 15, wherein the biological sample is a sample comprising nucleated cells.

17. The use of claim 15 or 16, wherein the biological sample is a blood sample, a semen sample or a hair sample.

18. The use of claim 15, wherein determining the genotype of the ABAC2 gene is performed by Polymerase Chain Reaction (PCR), hybridization, Southern blotting on a membrane, nuclease digestion, Restriction Fragment Length Polymorphism (RFLP), or direct sequencing techniques, or a combination thereof.

19. The use of claim 18, wherein determining the genotype of the ABAC2 gene comprises:

a. the DNA of an individual is extracted,

b. amplifying the isolated DNA using primers capable of amplifying a sequence corresponding to the rs908832 polymorphism of the ABCA2 gene,

c. determining the presence of at least one polymorphic allele of the rs908832 of the ABCA2 gene in the amplified DNA.

20. The use of claim 19, wherein the amplification step b comprises Polymerase Chain Reaction (PCR).

21. The use of claim 19, wherein the amplified DNA carrying the rs908832 polymorphic minor allele is distinguished from the amplified DNA not carrying the allele by a technique using the 5' nuclease activity of DNA polymerase i (taqman).

22. The use as claimed in claim 19, wherein the amplified DNA carrying the rs908832 polymorphic minor allele is distinguished from the amplified DNA not carrying the allele by Restriction Fragment Length Polymorphism (RFLP) analysis.

23. The use according to claim 22, wherein the restriction fragments have been obtained by digestion of the amplified DNA with restriction enzymes prior to migration with agarose gel, Southern blotting and hybridization on membranes.